Estrogen receptors and methods of use

ABSTRACT

The present invention provides isolated polypeptides having an amino acid sequence having at least 70% identity to SEQ ID NO:20, wherein the polypeptide has ER-α36 activity. The invention further provides methods for identifying agents that bind to such polypeptides, methods for detecting such polypeptides, and methods for altering the activity of such polypeptides. Also provided are antibodies that specifically bind to an amino acid sequence depicted at SEQ ID NO:1, or an immunogenic fragment thereof, and methods for making and using such antibodies.

CONTINUING APPLICATION DATA

This application is a divisional application of U.S. patent applicationSer. No. 13/177,523, filed on Jul. 6, 2011, which is a divisionalapplication of U.S. patent application Ser. No. 12/825,057, filed onJun. 28, 2010, and issued as U.S. Pat. No. 8,013,127, which is acontinuation application of Ser. No. 10/591,199, filed on Jun. 13, 2007,and issued as U.S. Pat. No. 7,745,230, which is National Stage Entry ofPCT/US05/07857, entitled ESTROGEN RECEPTORS AND METHODS OF USE, andclaims the benefit of U.S. Provisional Application Ser. No. 60/552,067,filed 10 Mar. 2004, and 60/643,469, filed 13 Jan. 2005, each of which isincorporated by reference herein.

GOVERNMENT FUNDING

The invention described herein was developed with support from theDepartment of Health and Human Services under Grant Number CA84328. TheU.S. Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Estrogen is a generic term for steroid compounds that are formed in theovary, the testis, and possibly the adrenal cortex. Examples ofestrogens and compounds having estrogen activity includediethylstilbestrol, fosfestrol, hexestrol, polyestradiol phosphate,broparoestrol, chlorotrianisene, dienestrol, diethylstilbestrol,methestrol, colpormon, equilenin, equilin, estradiol, estriol, estrone,ethinyl estradiol, mestranol, mexestrol, quinestradiol and quinestrol.Estrogens regulate diverse physiological processes in reproductivetissues and in mammary, cardiovascular, bone, liver, and brain tissues.Estrogens are also used in oral contraceptives. Other uses for estrogensinclude the relief of the discomforts of menopause, inhibition oflactation, and treatment of osteoporosis, threatened abortion, andvarious functional ovarian disorders. Anti-estrogens are used to treatmetastatic breast carcinoma and advanced prostate cancer.

The effects of estrogens are mediated via estrogen receptors. The firstestrogen receptor (ER) was cloned in 1986 (Green et. al., Nature,320:134 (1986) and Greene et. al., Science, 231:1150 (1986)). Until1995, it was assumed that there was only one estrogen receptorresponsible for all of the physiological and pharmacological effects ofnatural and synthetic estrogens and antiestrogens. However, in 1995, asecond estrogen receptor was cloned (Kuiper et. al., PNAS, 93:5925(1996)). The first estrogen receptor discovered is now called estrogenreceptor-alpha (ER-α) and the second estrogen receptor is calledestrogen receptor-beta (ER-β).

ER-α and ER-β share a common structural architecture (Zhang et. al.,FEBS Letters, 546:17 (2003) and Kong et. al., Biochem. Soc. Trans.,31:56 (2003)). Both are composed of three independent but interactingfunctional domains: the N-terminal A/B domain, the C or DNA-bindingdomain, and the D/E/F or ligand-binding domain (FIG. 1). The N-terminaldomain of ER-α encodes a ligand-independent activation function (AF-1),a region involved in interaction with co-activators, and transcriptionalactivation of target genes. The DNA-binding domain or C domain containsa two zinc-finger structure, which plays an important role in receptordimerization and binding to specific DNA sequences. The C-terminal D/E/Fdomain is a ligand-binding domain that mediates ligand binding, receptordimerization, nuclear translocation, and a ligand-dependenttransactivation function (AF-2). The relative contributions that bothAF-1 and AF-2 exert on transcriptional control vary in a cell-specificand DNA promoter-specific manner (Berry et. al., EMBO J., 9:2811 (1990)and Tzukerman et. al., Mol. Endocrin., 8:21 (1994)).

A 46-kDa ER-α isoform lacking the first 173 amino acids of thefull-length gene product of the ER-α gene (A/B or AF-1 domain) was shownto be derived from alternative splicing of the ER-α gene by skippingexon 1 (Flouriot et. al., EMBO 19:4688 (2000)). This alternativesplicing event generates an mRNA that has an AUG in a favorable Kozaksequence for translation initiation in frame with the remainder of theoriginal open reading frame. Therefore, this new isoform of ER-α wasnamed as ER-α46 and the original one was named ER-α66 (Flouriot et. al.,EMBO J., 19:4688 (2000)). ER-α46 forms homodimers and binds to anestrogen response element (ERE), and it can also form heterodimers withER-α66 (Flouriot et. al., EMBO J., 19:4688 (2000)). ER-α46 homodimersshow a higher affinity for an ERE than ER-α66 homodimers. Furthermore,the ER-α46/66 heterodimers form preferentially over the ER-α66homodimers and ER-α46 acts competitively to inhibit transactivationmediated by the AF-1 domain of liganded-ER-α66, but does not effectAF-2-dependent transactivation (Floutiot et. al., EMBO J., 19:4688(2000)). Therefore, it is thought that ER-α46 is a naturally occurringisoform of ER-α that regulates estrogen signaling mediated by the AF-1domain of ER-α66.

ER-α is expressed in approximately 15-30% of luminal epithelial cellsand not at all in any of the other cell types in the normal humanbreast. Dual label immunofluorescent techniques revealed thatER-α-expressing cells are separate from those labeled with proliferationmarkers in both normal human and rodent mammary glands (Clarke et. al.,Cancer Res., 57:4987 (1997)). ER-α expression is increased at the veryearliest stages of ductal hyperplasia and increases even more withincreasing atypia, such that most cells in atypical ductal hyperplasiasand in ductal cancer in situ of low and intermediate nuclear gradecontain the ER-α (Khan et. al., Cancer Res., 54:993 (1994) and Lawsonet. al., Lancet, 351:1787 (1994)). As ER-α expression increases, theinverse relationship between receptor expression and cell proliferationbecome dysregulated (Shoker et. al., Amer. Jour. Path., 155:1811(1999)). Approximately 70% of invasive breast carcinomas express theER-α and most of these tumors contain ER-α-positive proliferating cells(Clarke et. al., Cancer Res., 57:4987 (1997)).

Estrogen receptors are members of the nuclear receptor superfamily ofligand-activated transcription factors that control numerousphysiological processes. This control often occurs through theregulation of gene transcription (Katzenellenbogen and Katzenellenbogen,Breast Cancer Res., 2:335 (2000); Hull et al. J. Biol. Chem., 276:36869(2001); McDonnell and Norris, Science, 296:1642 (2002)). The estrogenreceptor utilizes multiple mechanisms to either activate or represstranscription of its target genes. These mechanisms include: (a) directinteraction of the ligand-occupied receptor with DNA at estrogenresponse elements followed by recruitment of transcriptional coregulatoror mediator complexes, (b) interaction of the ligand-occupied ER withother transcription factors such as AP-1 (Kushner at al., J. SteroidBiochem. Mol. Biol., 74:311 (2000)), Sp1 (Safe, Vitam. Horm. 62:231(2001)) or NF-κB (McKay and Cidlowski, Endocr. Rev., 20:435 (1999)), or(c) indirect modulation of gene transcription via sequestration ofgeneral/common transcriptional components (Hamish et al., Endocrinology,141:3403 (2000) and Speir et al., Circ. Res., 87:1006 (2000)). Inaddition, the ability of an estrogen receptor to regulate transcriptionthrough these various mechanisms appears to be cell-type specific,perhaps due to differences in the complement of transcriptionalcoregulatory factors available in each cell type (Cerillo et al., J.Steroid Biochem. Mol. Biol., 67:79 (1998): Evans et al., Circ. Res.,89:823 (2001); Maret et al., Endocrinology, 140:2876 (1999)). Also,transcriptional regulation is dependent upon the nature of the ligand,with various natural and synthetic selective estrogen receptormodulators acting as either estrogen receptor agonists or antagoniststhrough each of these various mechanisms (Shang and Brown, Science,295:2465 (2002); Katzenellenbogen and Katzenellenbogen, Science,295:2380 (2002); Margeat et al., J. Mol. Biol., 326:77 (2003); Dang etal, J. Biol. Chem., 278:962 (2003)).

Another signaling pathway mediated by estrogens, also known as a‘non-classic’, ‘non-genomic’ or ‘membrane signaling’ pathway, existsthat involves cytoplasmic proteins, growth factors and othermembrane-initiated signaling pathways (Segars et. al., Trends Endocrin.Met., 13:349 (2002)). Several intracellular signaling pathways have beenshown to cross-talk with rapid estrogen-initiated effects: the adenylatecyclase pathway (Aronica et. al., PNAS, 91:8517 (1994)), thephospholipase C pathway (Le Mellay et. al., J. Cell. Biochem., 75:138(1999)), the G-protein-coupled receptor-activated pathways (Razandi et.al., Mol. Endocrin., 13:307 (1999)) and the mitogen activated proteinkinase (MAPK) pathway (Watters et. al., Endocrinology, 138:4030 (1997)).However, all membrane forms described to date are related to ER-α butnot ER-β (Segars, et. al., Trends Endocrin. Met., 13:349 (2002)).

Estrogen signaling has been associated pathologically with an increasedrisk for breast and endometrial cancer (Summer and Fuqua, Semin. CancerBiol., 11:339 (2001); Turner et al., Endocr. Rev., 15:275 (1994); Farhatet al., FASEB J., 10:615 (1996); Beato et al., Cell, 83:851 (1995);Dobrzycka et al., Endo. Rel. Cancer, 10:517 (2003)). Consequently,estrogen receptors have been found to be essential in the initiation anddevelopment of most of these cancers. Current endocrine therapies forestrogen receptor-positive breast cancers are primarily designed totarget estrogen levels, estrogen receptor levels, or the activity ofestrogen and estrogen receptors. Use of a partial antiestrogen,tamoxifen, in the management of early-stage breast cancer has clearlydemonstrated an increase in both disease-free and overall survival. Inaddition, recent studies demonstrate that tamoxifen can be used as achemopreventive agent for hormone-dependent breast cancer. The majorconcerns of long-term therapy with tamoxifen are its uterotropiceffects, which result in an increase risk for endometrial cancer, andthe acquired clinical resistance to tamoxifen. This has led to theactive pursuit of better selective estrogen receptor modulators (SERM)that display the optimal agonistic or antagonistic activities in variousestrogen responsive target tissues.

Accordingly, what are needed are additional methods and materials thatcan be used to screen for agents that modulate estrogen signaling, aswell as methods and materials that can be used to modulate estrogensignaling.

SUMMARY OF THE INVENTION

The present invention provides an isolated antibody that specificallybinds to an amino acid sequence depicted at SEQ ID NO:1, or animmunogenic fragment thereof, preferably, an amino acid sequencedepicted at amino acids 13-27 of SEQ ID NO:1. The antibody may be amonoclonal antibody or a polyclonal antibody. Optionally, the antibodyis a humanized antibody. The antibody may be covalently attached to acompound such as, for instance, a chemotherapeutic agent or a detectablemarker such as a fluorescent marker. The antibody may be present in acomposition, and the composition may include a pharmaceuticallyacceptable carrier. Also provided are kits that include an antibody ofthe present invention.

The present invention also provides a method for making an antibody. Theantibody may be polyclonal or monoclonal. The method includesadministering to an animal a polypeptide having an amino acid sequencedepicted at SEQ ID NO:1, or an immunogenic fragment thereof, preferably,an amino acid sequence depicted at amino acids 13-27 of SEQ ID NO:1. Themethod further includes isolating antibody from the animal, wherein theisolated antibody specifically binds to the amino acid sequence. Thepolypeptide or immunogenic subunit thereof may be covalently attached toa carrier polypeptide. The isolating may include obtaining from theanimal a cell that produces the antibody, and making amonoclonal-antibody producing hybridoma using the cell. The inventionfurther includes a polyclonal antibody produced by the method and amonoclonal antibody produced by the method.

The invention is also directed to a cell including an exogenous codingregion, wherein the coding region encodes a polypeptide including SEQ IDNO:20. The coding region may encode a polypeptide having an amino acidsequence with at least 90% identity to SEQ ID NO:20, wherein thepolypeptide has ER-α36 activity. The coding region may be operablylinked to a constitutive promoter. The cell may be a eukaryotic cell ora prokaryotic cell. Also provided by the invention is a cell thatexpresses such polypeptides.

The present invention further provides a method for identifying an agentthat binds a polypeptide. The method includes combining a polypeptidethat includes an amino acid sequence depicted at SEQ ID NO:1, and anagent, and detecting the formation of a complex between the agent andthe polypeptide, detecting an alteration in the activity of thepolypeptide, or the combination thereof. The binding of the agent to thepolypeptide may be detected by directly detecting the binding of theagent to the polypeptide, detecting the binding of the agent to thepolypeptide using a competition binding assay, or the combinationthereof. Optionally, the method also includes determining whether theagent binds a polypeptide including SEQ ID NO:18.

Also provided by the present invention are methods for detectingpolypeptides. In one aspect, the method includes providing a cell,analyzing the cell for a polypeptide having ER-α36 activity and amolecular weight of 36 kDa as measured following electrophoresis on asodium dodecyl sulfate (SDS)-polyacrylamide gel, and determining whetherthe cell expresses the polypeptide. The cell may be ex vivo or in vivo.The cell may be, for instance, a tumor cell, such as a breast tumorcell. The analyzing may include contacting the cell with an antibodythat specifically binds to an amino acid sequence depicted at SEQ IDNO:1, or an immunogenic fragment thereof. The analyzing may includeamplifying an mRNA polynucleotide to form amplified polynucleotides. Theamplification includes contacting polynucleotides obtained from the cellwith a primer pair that will amplify an mRNA polynucleotide thatincludes SEQ ID NO:22 or SEQ ID NO:25, or the combination thereof,wherein the presence of amplified polynucleotides indicates the cellexpresses the polypeptide. One primer of the primer pair may be chosenfrom nucleotides of SEQ ID NO:22, nucleotides complementary tonucleotides of SEQ ID NO:25, or the combination thereof.

The invention also provides a method for inhibiting ER-α36 activity of acell. The method includes contacting a cell expressing a polypeptidehaving an amino acid sequence depicted at SEQ ID NO:1 with a compoundthat inhibits ER-α36 activity. Such a compound may be an antibody thatspecifically binds to a polypeptide having an amino acid sequencedepicted at amino acids 13-27 of SEQ ID NO:1. The cell may be in vivo orex vivo, and optionally may be ER-α66 negative. ER-α46 negative, or thecombination thereof. In some aspects the compound is not ananti-estrogen.

Further provided by the present invention is an isolated polypeptidethat includes an amino acid sequence depicted at amino acids 13-27 ofSEQ ID NO:1, preferably an amino acid sequence depicted SEQ ID NO:1,more preferably, an amino acid sequence depicted at SEQ ID NO:20. Inanother aspect, the isolated polynucleotide has at least 70% identity toSEQ ID NO:20, wherein the polypeptide has ER-α36 activity. The presentinvention also includes an immunogenic fragment of SEQ ID NO:1.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims. Unlessotherwise specified, “a,” “an.” “the,” and “at least one” are usedinterchangeably and mean one or more than one.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the domain structure representation of Human estrogenreceptor-alpha (ER-α) isoforms. Domains (labeled A-F), amino acidsequence numbering, AF-1 and AF-2, the DNA binding domain, theligand-binding domain, and the dimerization domain are shown. Thephosphorylation sites and function of each domain are also indicated.

FIG. 2 is a schematic demonstrating the possible crosstalk between themembrane and genomic signaling pathways of ER-α. Cav-1 representscaveolin-1, ER-α, estrogen receptor-alpha; RTK, a receptor tyrosinekinase; Ras, Ras oncogene; Mek, MAP/ERK kinase; MAPK, a mitogenactivated protein kinase; PI3K, phosphoinositol-triphosphate kinase;AKT, protein kinase 13; PDK1, phosphoinositol-dependent protein kinase;RSK, p90 ribosome S6 kinase.

FIG. 3 is a picture showing that pRET-infected MCF10A cells grow a bigcolony in soft-agar in the presence of estradiol (E2). The ST1 cloneshows an accelerated growth in E2-containing soft-agar. MCF7 and MCF10Acells are included as a positive and negative control, receptively.

FIG. 4 is a Western blot showing downregulation of Caveolin-1 (Cav-1)expression in pRET-infected MCF10A cells. Equal amounts of totalcellular extracts from various cell lines were analyzed by Western blotusing a rabbit anti-Cav-1 antibody (N20). The position of Cav-1 isindicated by an arrow and the cell extract analyzed in each lane isindicted above each lane.

FIG. 5 is a western blot showing upregulation of ER-α expression inpRET-infected MCF10A cells. Equal amounts of total cellular extractsfrom various cell lines were analyzed by Western blot using antibodiesagainst ER-α (H222) and ER-β. The positions of ER-α and ER-β areindicated by arrows and the cell extract analyzed in each lane isindicted above each lane.

FIG. 6 is a Western blot showing activation of ERK1/2 phosphorylation inpRET-infected MCF10A cells. Equal amounts of total cellular extractsfrom the cell lines were analyzed by Western blot using antibodiesagainst ERK1/2 and phosphorylated ERK1/2.

FIG. 7 is a Western blot showing the existence of three ER-α proteins inCav-1 haploinsufficient cells, ST1 and ST3, and MCF7 breast cancercells. Equal amounts of total cellular extracts from the cell lines wereanalyzed by Western blot using the H222 antibody against ER-α. Thepositions of ER-α66, ER-α46 and ER-α36 are indicated by arrows and thecell extract analyzed in each lane is indicted above each lane.

FIG. 8 illustrates the genomic organization of the human ER-α gene. Thelocation of multiple promoters are shown as arrows. The translationstart and stop sites are indicated as AUG and UGA. The exons are shownas numbered boxes. Intron 1 is also shown with the exon 1′ in a box. Thelower panel shows the mRNA structure of ER-α isoforms. Poly A sites areindicated by AAA.

FIG. 9 is a picture of an agarose gel showing the isolation of cDNAencoding the open-reading frame of ER-α36 by PCR. The position of thecDNA in the gel is indicated by an arrow.

FIG. 10 shows the predicted amino acid sequence of the ER-α36open-reading frame. The amino acid positions are indicated by numbers onthe left side of the amino acid sequence (SEQ ID NO:20). The last 27amino acids that are unique to ER-α36 are underlined.

FIG. 11 shows a Western blot analysis of ER-α66, ER-α46 and ER-α36. Thelanes marked ER-α66, ER-α46 and ER-α36 represent separated cultures ofHEK 293 cells that were transfected with expression plasmids encodingthe indicated estrogen receptor isoform, and lysed two days after beingtransfected. The lysate of each transfectant was immunodetected with ananti-ER-α antibody (H222). The cell extracts from MCF7 cells used as apositive control. The positions of ER-α66, ER-α46 and ER-α36 areindicated by arrows.

FIG. 12 shows (a) the DNA sequence of the 5′ flanking sequence (SEQ IDNO:22) of the gene that encodes ER-α36 and which includes the ER-α36promoter, and (b) the DNA sequence of the 3′ flanking sequence (SEQ IDNO:25) of the gene that encodes ER-α36 and which includes thenucleotides encoded by exon 9. In the 5′ flanking sequence the putativetranscription binding sites are underlined and the proteins that bind tothe nucleic acid sequence are also indicated. The initiation site of thecDNA is also indicated by an arrow.

FIG. 13 shows Northern blot analysis of ER-α36 in different breastcancer cells MCF10A, T47D, MCF7, and MDA-MB-231. The positions of ER-α36and actin are indicated by arrows.

FIG. 14 shows inhibition by ER-α36 of the transcriptionaltransactivation activities mediated by the AF-1 and AF-2 domains ofER-α66 and ER-β. (+E2), cells treated with E2; (−E2), cells not treatedwith E2.

FIG. 15 shows ER-α36 mediates membrane-initiated MAPK kinase pathwaystimulated by E2. (a) Western blot shows treatment of ER36-293 cellswith estradiol-17β (E2β) induces rapid phosphorylation of Mek1/2 andERK1/2. P-Mek1/2 and P-ERK1/2, phosphorylated forms of Mek1/2 andERK1/2, respectively. (b) Serum but not E2β induces phosphorylation ofERK1/2 in control vector-293 cells. P-ERK1/2, phosphorylated form ofERK1/2. (c) Different estrogens and antiestrogens induce rapidphosphorylation of ERK1/2 in ER36-293 cells. P-ERK1/2, phosphorylatedform of ERK1/2. (d) Tamoxifen treatment constitutively stimulates ERK1/2phosphorylation in ER36-293 cells. P-ERK1/2, phosphorylated form ofERK1/2.

FIG. 16 shows ER-α36 mediates E2β induced MAPK kinase nuclear signalingand stimulates cell growth. (a) Effects of E2β on MAPK kinase nuclearsignaling. ER36-293 and control vector-293 cells were transientlytransfected with 5×Gal4-LUC, a luciferase reporter plasmid containingfive Gal4 DNA binding sites, and Gal-ELK expression vector containing anELK transcriptional activation domain fused with the Gal4 DNA bindingdomain (Upper panel). After transfection, the cell culture wasmaintained in estrogen free medium for 36 hours before E21 (1 nM or 10nM) was added for 12 hours. Luciferase activities with standarddeviation are representative of more than three experiments performed induplicates. (b) E2β and anti-estrogens stimulate growth of ER36-293cells. Absorbance data at 490 nm are shown. Results of more than fiveindependent experiments have were averaged; the mean and SEM are shown.The statistical significance of these results was also evaluated bypaired t-test. P-values were <0.001 for ER36-293 and vector-293 cells.

FIG. 17 shows ER-α36 is mainly a membrane-based estrogen receptor. (a)Western blot analysis of expression of ER-α36 in different establishedbreast cancer cell lines. The same blot was stripped and probed with ananti-actin antibody to ensure equal loading. (b) Subcellularlocalization of ER-α36 in ER-06 transfected 293 cells. Immunoblot ofER-α36 in different subcellular fractions with the ER-α36 specificantibody. W, whole cell lysate; PM, plasma membrane: C, cytosol; N,nucleus. Subcellular fraction purity was examined by immunoblottingvarious protein markers for the plasma membrane, cytosol, nucleus andGolgi. 5′NT, 5′ nucleotidase; D4-GDI, GDP dissociation inhibitor;mSin3A, a component of histone remodeling complex; COPB, β coat protein.

FIG. 18 shows that E2β promotes growth of ER-α66 negative breast cancercells, MDA-MB-231, in soft agar. MDA-MB-231 cells were grown on softagar for three weeks in the absence of E2β(0), and in the presence of 10nM E2β (E2) 10 nM E2β and 10 nM Tamoxifen (E2+TAM) and 10 nM Tamoxifenalone (TAM).

FIG. 19 shows that E2β induces membrane initiated estrogen signaling inER-α66 negative breast cancer cells MDA-MB-231. Treatment of MDA-MB-231cells with estradiol-17β (E2β) induced rapid phosphorylation of ERK1/2.Cells were treated with E2β (10 nM) for different time points, lysed andanalyzed with Western blot using phosphorylation dependent andindependent antibodies.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

It has been discovered that downregulation of the Caveolin-1 (Cav-1)system constitutively activates the mitogen activated protein kinase(MAPK) pathway, activates expression of estrogen receptor-alpha (ER-α),and triggers positive estrogen signaling. This discovery has, for thefirst time, provided a clear link between activated MAPK signaling andmammary tumorigenesis, especially breast cancer progression that isstimulated by estrogens. This discovery strongly suggests that Cav-1plays an important role in maintaining normal growth of mammaryepithelial cells by coordinating the cross-talk between the MAPK andestrogen signaling pathways, and its downregulation may contribute todysregulation of these two important pathways which eventually lead tomammary tumorigenesis. A schematic of the estrogen signaling pathway andthe MAPK signaling pathway is presented in FIG. 2.

An estrogen receptor-alpha isoform has also been identified and cloned.This 36-kDa isoform (ER-α36) of estrogen receptor-alpha is generatedfrom a promoter located in the first intron of the original 66-kDa ER-α(ER-α66) gene. ER-α36 differs from ER-α66 because it lacks bothtranscriptional activation domains (AF-1 and AF-2) but retains theDNA-binding, dimerization and most of the ligand-binding domains. Thestructure of ER-α36 indicates that ER-α36 is a regulator of estrogensignaling. ER-α36 may also mediate the membrane effects of estrogensignaling as it is primarily expressed on the plasma membrane, and alsoin cytosol and nucleus.

ER-β has been proposed as a constitutive regulator of ER-α66 mediatedestrogen signaling. The finding that ER-α46 lacking the AF-1 domain candimerize to ER-α66 and inhibit the transactivation activity mediated bythe AF-1 domain of ER-α66 indicates that ER-α46 plays a regulatory rolein the functional activity mediated by the AF-1 domain of ER-α66. ER-α36lacks both AF-1 and AF-2 domains. Thus, it is thought that ER-α36inhibits biological functions mediated by both AF-1 and AF-2 of ER-α66,and AF-2 mediated functions of ER-α46 as well. With regulation mediatedby ER-α36 and ER-α46, both of which might be expressed at differentlevels in different tissues, ER-α66 may function differently indifferent target tissues. Such a mechanism is thought to provide anexplanation for the pleiotrophic roles of estrogen signaling indifferent biological processes.

Polypeptides and Peptidomimetics of the Invention

The invention provides polypeptides. As used herein, the term“polypeptide” refers broadly to a polymer of two or more amino acidsjoined together by peptide bonds. The terms peptide, oligopeptide, andprotein are all included within the definition of polypeptide and theseterms are used interchangeably. It should be understood that these termsdo not connote a specific length of a polymer of amino acids, nor arethey intended to imply or distinguish whether the polypeptide isproduced using recombinant techniques, chemical or enzymatic synthesis,or is naturally occurring. Numerous examples of polypeptides that arewithin the scope of the invention are disclosed and described herein. Inthe case of a polypeptide or polynucleotide that is naturally occurring,it is preferred that such polypeptide or polynucleotide be isolated and,optionally, purified. An “isolated” polypeptide or polynucleotide is onethat is separate and discrete from its natural environment. A “purified”polypeptide or polynucleotide is one that is at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated. Polypeptides and nucleotidesthat are produced outside the organism in which they naturally occur,e.g., through chemical or recombinant means, are considered to beisolated and purified by definition, since they were never present in anatural environment. An “exogenous polypeptide” refers to a foreignpolypeptide, i.e., a polypeptide that is not normally present in a cell,or a polypeptide that is normally present in a cell but has beenintroduced into the cell by experimental procedure, e.g., byintroduction of a polynucleotide encoding the polypeptide.

The polypeptides of the present invention may be biologically active.Such biological activity is referred to herein as “ER-α36 activity.” Aexample of a bioassay that can be used to determine if a polypeptide ofthe invention is biologically active involves contacting a cell thatexpresses this polypeptide with an estrogen or anti-estrogen anddetermining if activities of the MAPK pathway are increased or decreasedin the presence of the estrogen or anti-estrogen, when compared to theMAPK activities in a control cell that was not expressing thepolypeptide of the invention. Preferably, the MAPK activities arephosphorylation of ERK 1/2 and Mek 1/2, and preferably thephosphorylation of ERK 1/2 induced by a polypeptide of the presentinvention is not decreased in the presence of an anti-estrogen.Preferably, ER-α36 activity is membrane initiated. ER-α36 activity maybe measured by exposing a cell expressing a polypeptide that may haveER-α36 activity to a different ligands. Examples of ligands that can beused include, but are not limited to, estrone (E1), 17α-estradiol (E2α),17β-estradiol (E2β), estriol (E3), estetrol (E4), or an estrogenattached to a membrane impermeable molecule, for instance, bovine serumalbumin (BSA). Generally, when the ER-α36 activity to be measured is tobe limited to membrane initiated ER-α36 activity, an estrogen attachedto a membrane impermeable molecule is used. The amount of estrogen usedcan vary, and is preferably in the range of between 1 nM and 10 nM. Thecell exposed to the estrogen is preferably a quiescent cell. Theexposure is allowed to occur for between 5 and 90 minutes, the cell isthen lysed, and the polypeptides present in the cell are resolved bySDS-polyacrylamide gel electrophoresis. After transfer of the resolvedpolypeptides to a membrane, antibodies against the non-phosphorylatedand phosphorylated forms of ERK 1/2 and Mek 1/2 are used to evaluateactivation of the MAPK pathway. Optionally, an anti-estrogen, such asTamoxifen, 40H-Tamoxifen, or ICI-182,780, may be included to determineif the phosphorylation of ERK 1/2 is insensitive to anti-estrogens.

The invention provides a polypeptide having the amino acid sequencedepicted in SEQ ID NO:20. This polypeptide, and related polypeptides asdescribed herein, are also referred to herein as ER-α36, ER-α36 isoform,and ER receptor α36-subunit. As shown in FIG. 1, the ER-α36 isoformlacks amino-terminal amino acid residues 1-183, carboxyl-terminal aminoacid residues 430-595, and has an addition of 27 amino acid residues toits C- terminus when compared to the ER-α66 isoform (see Table 1).Estrogen receptor alpha isomers include ER-α36, ER-α46, ER-α66. Estrogenreceptor beta isomers include ER-β. The present invention also providesestrogen receptors that include an ER-α36 isoform. Without intending tobe limiting, the ER-α36 isoform is believed to modulate the response ofa cell to estrogen through regulation of estrogen receptor function byforming a dimer with ER-α66, ER-α46 or ER-β. Further, ER-α36 is thoughtto lack activation factor 1 (AF-1) and activation factor 2 (AF-2)activity, and thus lacks intrinsic transcription activity. However,ER-α36 is thought to retain an intact dimerization domain that allowsER-α36 to dimerize with an ER-α46, ER-α66 or ER-β. This interaction isthought to allow ER-α36 to modulate the activity of ER-α46, ER-α66 andER-β containing estrogen receptors.

TABLE 1 Amino acid and nucleotide sequences SEQ ID NO and DescriptionAmino acid and nucleotide Sequences SEQ ID NO: 18,MTMTLHTKASGMALLHQIQGNELEPLNRPQLKIPLERPL ER-α66,GEVYLDSSKPAVYNYPEGAAYEFNAAAAANAQVYGQTGL AccessionPYGPGSEAAAFGSNGLGGFPPLNSVSPSPLMLLHPPPQL NumbersSPFLQPHGQQVPYYLENEPSGYTVREAGPPAFYRPNSDN M12674,RRQGGRERLASTNDKGSMAMESAKETRYCAVCNDYASGY AAA52399HYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKCYEVGMMKGGIRKDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTELSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYIT GEAEGFPATV SEQ ID NO: 19,ATGACCATGACCCTCCACACCAAAGCATCTGGGATGGCCCTACTGCATCA ER-α66,GATCCAAGGGAACGAGCTGGAGCCCCTGAACCGTCCGCAGCTCAAGATCC AccessionCCCTGGAGCGGCCCCTGGGCGAGGTGTACCTGGACAGCAGCAAGCCCGCC NumberGTGTACAACTACCCCGAGGGCGCCGCCTACGAGTTCAACGCCGCGGCCGC M12674,CGCCAACGCGCAGGTCTACGGTCAGACCGGCCTCCCCTACGGCCCCGGGT AY425004CTGAGGCTGCGGCGTTCGGCTCCAACGGCCTGGGGGGTTTCCCCCCACTCAACAGCGTGTCTCCGAGCCCGCTGATGCTACTGCACCCGCCGCCGCAGCTGTCGCCTTTCCTGCAGCCCCACGGCCAGCAGGTGCCCTACTACCTGGAGAACGAGCCCAGCGGCTACACGGTGCGCGAGGCCGGCCCGCCGGCATTCTACAGGCCAAATTCAGATAATCGACGCCAGGGTGGCAGAGAAAGATTGGCCAGTACCAATGACAAGGGAAGTATGGCTATGGAATCTGCCAAGGAGACTCGCTACTGTGCAGTGTGCAATGACTATGCTTCAGGCTACCATTATGGAGTCTGGTCCTGTGAGGGCTGCAAGGCCTTCTTCAAGAGAAGTATTCAAGGACATAACGACTATATGTGTCCAGCCACCAACCAGTGCACCATTGATAAAAACAGGAGGAAGAGCTGCCAGGCCTGCCGGCTCCGCAAATGCTACGAAGTGGGAATGATGAAAGGTGGGATACGAAAAGACCGAAGAGGAGGGAGAATGTTGAAACACAAGCGCCAGAGAGATGATGGGGAGGGCAGGGGTGAAGTGGGGTCTGCTGGAGACATGAGAGCTGCCAACCTTTGGCCAAGCCCGCTCATGATCAAACGCTCTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTCAGTGCCTTGTTGGATGCTGAGCCCCCCATACTCTATTCCGAGTATGATCCTACCAGACCCTTCAGTGAAGCTTCGATGATGGGCTTACTGACCAACCTGGCAGACAGGGAGCTGGTTCACATGATCAACTGGGCGAAGAGGGTGCCAGGCTTTGTGGATTTGACCCTCCATGATCAGGTCCACCTTCTAGAATGTGCCTGGCTAGAGATCCTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCACCCAGTGAAGCTACTGTTTGCTCCTAACTTGCTCTTGGACAGGAACCAGGGAAAATGTGTAGAGGGCATGGTGGAGATCTTCGACATGCTGCTGGCTACATCATCTCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGTTTGTGTGCCTCAAATCTATTATTTTGCTTAATTCTGGAGTGTACACATTTCTGTCCAGCACCCTGAAGTCTCTGGAAGAGAAGGACCATATCCACCGAGTCCTGGACAAGATCACAGACACTTTGATCCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCGGCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCAGGCACATGAGTAACAAAGGCATGGAGCATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGACCTGCTGCTGGAGATGCTGGACGCCCACCGCCTACATGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAGGAGACGGACCAAAGCCACTTGGCCACTGCGGGCTCTACTTCATCGCATTCCTTGCAAAAGTATTACATCACGGGGGAGGCAGAGGGTTTCCCTGCCACAGTCTGA SEQ ID NO: 21,ATGGCTATGGAATCTGCCAAGGAGACTCGCTACTGTGCAGTGTGCAATGA ER-α36,CTATGCTTCAGGCTACCATTATGGAGTCTGGTCCTGTGAGGGCTGCAAGG NucleotidesCCTTCTTCAAGAGAAGTATTCAAGGACATAACGACTATATGTGTCCAGCC 234-1166 ofACCAACCAGTGCACCATTGATAAAAACAGGAGGAAGAGCTGCCAGGCCTG AccessionCCGGCTCCGCAAATGCTACGAAGTGGGAATGATGAAAGGTGGGATACGAA NumberAAGACCGAAGAGGAGGGAGAATGTTGAAACACAAGCGCCAGAGAGATGAT BX640939GGGGAGGGCAGGGGTGAAGTGGGGTCTGCTGGAGACATGAGAGCTGCCAACCTTTGGCCAAGCCCGCTCATGATCAAACGCTCTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTCAGTGCCTTGTTGGATGCTGAGCCCCCCATACTCTATTCCGAGTATGATCCTACCAGACCCTTCAGTGAAGCTTCGATGATGGGCTTACTGACCAACCTGGCAGACAGGGAGCTGGTTCACATGATCAACTGGGCGAAGAGGGTGCCAGGCTTTGTGGATTTGACCCTCCATGATCAGGTCCACCTTCTAGAATGTGCCTGGCTAGAGATCCTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCACCCAGGGAAGCTACTGTTTGCTCCTAACTTGCTCTTGGACAGGAACCAGGGAAAATGTGTAGAGGGCATGGTGGAGATCTTCGACATGCTGCTGGCTACATCATCTCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGTTTGTGTGCCTCAAATCTATTCTTTTGCTTAATTCTGGTATCTCACATGTAGAAGCAAAGAAGAGAATCCTGAACTTGCATCCTAAAATATTTGGAAACAAGTGGTTTCCTCGTGTCTAA

Polypeptides of the present invention include polypeptides having anamino acid sequence that is at least 70% identical to SEQ ID NO:20. Suchpolypeptides include those having an amino acid sequence that is atleast single unit percentages greater than 70% identical to SEQ IDNO:20, for example 71%, 72%, 73% identity, and so on to 100% identity toSEQ ID NO:20. Preferably, the polypeptide includes those having an aminoacid sequence that is, in increasing order of preference, at least about80% identity, at least about 90% identity, or at least about 95%identity to SEQ ID NO:20. Preferably the polypeptide is biologicallyactive. Preferably the polypeptide has a molecular weight of 36 kDa asmeasured following electrophoresis on a sodium dodecyl sulfate(SDS)-polyacrylamide gel. Typically, residues involved inphosphorylation of ER-α66, e.g., S236, K302, and K303 are conserved, asare those residues involved in the function of the DNA binding domain,ligand binding domain, and dimerization domains of ER-α66. Residues thatfunction in DNA binding, ligand binding, and/or dimerization are knownin the art.

Percent identity between two polypeptide sequences is generallydetermined by aligning the residues of the two amino acid sequences tooptimize the number of identical amino acids along the lengths of theirsequences; gaps in either or both sequences are permitted in making thealignment in order to optimize the number of identical amino acids,although the amino acids in each sequence must nonetheless remain intheir proper order. Preferably, two amino acid sequences are comparedusing the Blastp program, version 2.0.9, of the BLAST 2 searchalgorithm, as described by Tatusova et al. (FEMS Microbiol. Lett., 174,247-250 (1999)), and available on the world wide web athttp://www.ncbi.nlm.nih.gov/blast/bl2seq/b12.html. Preferably, thedefault values for all BLAST 2 search parameters are used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapx_dropoff=50, expect=10, wordsize=3, and optionally, filter on. In thecomparison of two amino acid sequences using the BLAST search algorithm,structural similarity is referred to as “identity.”

Polypeptides that are fragments of the ER-α36 estrogen receptor isoformare also provided by the invention. Preferably, a fragment isimmunogenic. In some aspects, a fragment has ER-α36 activity. An exampleof an immunogenic fragment is the amino acid sequence depicted at aminoacids 13-27 of SEQ ID NO:1, more preferably, 1-27 of SEQ ID NO:1. Suchfragments are useful for preparing antibodies that specifically bind tothe ER-α36 estrogen receptor isoform. Examples of fragments include anestrogen receptor isoform that has been truncated at either theN-terminus, or the C-terminus, or both, by one or more amino acids, aslong as the fragment contains at least 5 contiguous amino acids, morepreferably at least 7 contiguous amino acids, even more preferably atleast contiguous 10 amino acids, and most preferably at least contiguous12 amino acids.

The invention provides fusion polypeptides having a carrier polypeptidecoupled to a polypeptide of the invention. A carrier polypeptide may beused to increase or decrease the solubility of a fusion polypeptide. Thecarrier polypeptide may also be used to increase the immunogenicity ofthe fusion polypeptide to increase production of antibodies that bind toa polypeptide of the invention. For example, a carrier polypeptide maybe fused to a fragment of a polypeptide of the present invention tofacilitate production of antibodies that specifically bind ER-α36. Anexample of such a fragment is a polypeptide having amino acids 13-27 SEQID NO:1. The invention is not limited by the types of carrierpolypeptides used to create fusion polypeptides of the invention.Examples of carrier polypeptides include keyhole limpet hemacyanin,bovine serum albumin, ovalbumin, mouse serum albumin, rabbit serumalbumin, and the like. The carrier polypeptides may also be used toprovide for the separation or detection of a fusion polypeptide.Examples of such carrier proteins include glutathione-S-transferase,maltose-binding protein, chitin-binding protein, and polypeptides havingthe following amino acid sequences: QFFGLM (SEQ ID NO:2), EQKLISEEDL(SEQ ID NO:3), KAEDESS (SEQ ID NO:4), YPYDVPDYA (SEQ ID NO:5), DYKDDDDK(SEQ ID NO:6), YTDIEMNRLGK (SEQ ID NO:7), MASMTGGQQMG (SEQ ID NO:8),DTYRYI (SEQ ID NO:9), TDFYLK (SEQ ID NO:10), HHHHHH (SEQ ID NO:11), HPOL(SEQ ID NO:12), QYPALT (SEQ ID NO:13), QRQYGDVFKGD (SEQ ID NO:14),EYMPME (SEQ ID NO:15), EFMPME (SEQ ID NO:16), and RYIRS (SEQ ID NO:17).Accordingly, a fusion polypeptide can be detected or isolated byinteraction with other components that bind to the carrier polypeptideportion of the fusion polypeptide. For example, a fusion polypeptidehaving avidin as a carrier polypeptide can be detected or separated withbiotin through use of known methods. A carrier polypeptide may also beused to cause the fusion polypeptide to form an inclusion body uponexpression within a cell. A carrier polypeptide can also be an exportsignal that causes export of a fusion polypeptide out of a cell, ordirects a fusion polypeptide to a compartment within a cell, such as theperiplasm.

The invention also provides two or more polypeptides of the inventionthat are continuously linked into a single amino acid chain. Such apolypeptide is referred to herein as a polypeptide. The polypeptides canbe connected by linkers (see Stahl et al., U.S. Pat. No. 6,558,924).Such a polyprotein can be isolated and then cleaved to producepolypeptides or coupled polypeptides of the invention. The polyproteincan be cleaved through use of numerous methods, such as chemical orprotease cleavage. Accordingly, linkers can be designed to be cleaved byspecific proteases or chemicals. Examples of compounds that can be usedto cleave polyproteins of the invention include chemicals and enzymes.Examples of chemicals include cyanogen bromide, formic acid and heat,hydroxylamine and heat, iodosobenzoicacid-2-(2-nitrophenyl)-3-methyl-3-bromoindole-nine in acetic acid, andthe like. Examples of enzymes include Ala-64 subtilisin, clostripain,collagenase, enterokinase, factor Xa, renin, α-thrombin, trypsin,chymotrypsin, tobacco etch virus protease, and the like. Polyproteinsmay be used to increase the production efficiency of the polypeptides ofthe invention. Methods to produce polyproteins are known in the art (seeCoolidge et al., U.S. Pat. No. 6,127,150).

The polypeptides of the invention include analogs that have beenmodified by the addition, substitution, or deletion of one or morecontiguous or noncontiguous amino acids, or that have been chemically orenzymatically modified, e.g., by attachment of a reporter group, by anN-terminal, C-terminal or other functional group modification orderivatization, or by cyclization, as long as the analog retainsbiological activity or is able to stimulate the production of antibodiesthat bind to ER-α36. An analog can thus include additional amino acidsat one or both of the termini of a polypeptide. Preferably, an analog isimmunogenic, more preferably, an analog is immunogenic and has ER-α36activity. In some aspects, the invention provides polypeptides that arenot analogs.

Substitutes for an amino acid in the polypeptides of the invention arepreferably conservative substitutions, which are selected from othermembers of the class to which the amino acid belongs. For example, it iswell-known in the art of protein biochemistry that an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic (such as charge, hydrophobicity and hydrophilicity) cangenerally be substituted for another amino acid without substantiallyaltering the structure of a polypeptide. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and tyrosine. Polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine, asparagineand glutamine. The positively charged (basic) amino acids includearginine, lysine and histidine. The negatively charged (acidic) aminoacids include aspartic acid and glutamic acid. Examples of preferredconservative substitutions include Lys for Arg and vice versa tomaintain a positive charge; Glu for Asp and vice versa to maintain anegative charge; Ser for Thr so that a free —OH is maintained; and Glnfor Asn to maintain a free NH₂. Related amino acids (such as3-hydroxyproline, 4-hydroxyproline, homocysteine, 2-aminoadipic acid,2-aminopimelic acid, γ-carboxyglutamicacid, β-carboxyaspartic acid),amino acid amides (ornithine, homoarginine, N-methyl lysine, dimethyllysine, trimethyl lysine, 2,3-diaminopropionic acid, 2,4-diaminobutyricacid, homoarginine, sarcosine and hydroxylysine) and substitutedphenylalanines, norleucine, norvaline, 2-aminooctanoic acid,2-aminoheptanoic acid, statine, β-valine, naphthylalanines,tetrahydroisoquinoline-3-carboxylic acid, and halogenated tyrosines maybe exchanged for a like amino acid.

The invention provides peptidomimetics of the polypeptides of theinvention. A peptidomimetic describes a polypeptide in which at leastone of the peptide bonds has been replaced with a non-peptide bond, suchas those commonly used in the pharmaceutical industry as non-peptidedrugs, with properties analogous to those of the template polypeptide.(Fauchere, J., Adv. Drug Res., 15: 29 (1986), Evans et al., J. Med.Chem., 30:1229 (1987), and Janda et al., U.S. Pat. No. 6,664,372).Peptidomimetics are structurally similar to polypeptides having peptidebonds, but have one or more peptide linkages optionally replaced by alinkage such as, —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans),—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methods known in the art.Advantages of peptidomimetics over natural polypeptide embodiments mayinclude more economical production, greater chemical stability, alteredspecificity and enhanced pharmacological properties such as half-life,absorption, potency and efficacy.

Substitution of one or more amino acids within a polypeptide or apeptidomimetic with a D-amino acid of the same type (e.g., D-lysine inplace of L-lysine) may be used to generate polypeptides andpeptidomimetics that are, for instance, more stable and more resistantto endogenous proteases.

Polypeptides and peptidomimetics of the invention can be modified for invivo use by the addition, at the amino-terminus and/or thecarboxyl-terminus, of a blocking agent to decrease degradation in vivo.This can be useful in those situations in which the polypeptide terminitend to be degraded by proteases in vivo. Such blocking agents caninclude, without limitation, additional related or unrelated peptidesequences that can be attached to the amino and/or carboxyl terminalresidues of the polypeptide or peptidomimetic of the invention. This canbe done during chemical synthesis, or by recombinant DNA technology bymethods familiar to artisans of average skill. Alternatively, blockingagents such as pyroglutamic acid, or other molecules known in the art,can be attached to the amino and/or carboxyl terminal residues, or theamino group at the amino terminus or carboxyl group at the carboxylterminus can be replaced with a different moiety. Accordingly, theinvention provides polypeptides and peptidomimetics that are blocked andthe amino tee minus, the carboxyl terminus, or the combination thereof.

Polypeptides of the invention can be produced on a small or large scalethrough use of numerous expression systems that include, but are notlimited to, cells or microorganisms that are transformed with arecombinant vector into which a polynucleotide of the invention has beeninserted. Such recombinant vectors and methods for their use aredescribed below. These vectors can be used to transform a variety oforganisms. Examples of such organisms include bacteria (for example, E.coli or B. subtilis); yeast (for example, Saccharomyces and Pichia);insects (for example, baculovirus); plants; or mammalian cells (forexample, COS, CHO, BHK, 293, VERO, HeLa, MDCK, W138, and NIH 3T3 cells).Also useful as host cells are primary or secondary cells obtaineddirectly from a mammal that are transfected with a vector.

Synthetic methods may also be used to produce polypeptides andpeptidomimetics of the invention. Such methods are known in the art andare routine. For instance, the solid phase peptide synthetic method isan established and widely used method. Polypeptides can be readilypurified by fractionation on immunoaffinity or ion-exchange columns;ethanol precipitation; reverse phase HPLC; chromatography on silica oron an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; ligand affinity chromatography, and the like.Polypeptides can also be readily purified through binding of a fusionpolypeptide to separation media, followed by cleavage of the fusionpolypeptide to release a purified polypeptide. For example, a fusionpolypeptide that includes a factor Xa cleavage site between thepolypeptide and the carrier polypeptide can be created. The fusionpolypeptide can be bound to an affinity column to which the carrierpolypeptide portion of the fusion polypeptide binds. The fusionpolypeptide can then be cleaved with factor Xa to release thepolypeptide. Such a system has been used in conjunction with a factor Xaremoval kit for purification of the polypeptides of the invention.

Polynucleotides

The invention provides polynucleotides that encode the polypeptides ofthe invention. The term “polynucleotide” refers broadly to a polymer oftwo or more nucleotides covalently linked in a 5′ to 3′ orientation. Apolynucleotide may include nucleotide sequences having differentfunctions, including for instance coding sequences, and non-codingsequences such as regulatory sequences. Coding sequence, non-codingsequence, and regulatory sequence are defined below. The terms nucleicacid, nucleic acid molecule, and oligonucleotide and protein includedwithin the definition of polynucleotide and these terms are usedinterchangeably. It should be understood that these terms do not connotea specific length of a polymer of nucleotides, nor are they intended toimply or distinguish whether the polynucleotide is produced usingrecombinant techniques, chemical or enzymatic synthesis, or is naturallyoccurring.

Polynucleotides can be single-stranded or double-stranded, and thesequence of the second, complementary strand is dictated by the sequenceof the first strand. The term “polynucleotide” is therefore to bebroadly interpreted as encompassing a single stranded nucleic acidpolymer, its complement, and the duplex formed thereby.“Complementarity” of polynucleotides refers to the ability of twosingle-stranded polynucleotides to base pair with each other, in whichan adenine on one polynucleotide will base pair with a thymidine (oruracil, in the case of RNA) on the other, and a cytidine on onepolynucleotide will base pair with a guanine on the other. Twopolynucleotides are complementary to each other when a nucleotidesequence in one polynucleotide can base pair with a nucleotide sequencein a second polynucleotide. For instance, 5′-ATGC and 5′-GCAT are fullycomplementary, as are 5′-GCTA and 5′-TAGC.

An example of a polynucleotide of the present invention is SEQ ID NO:21(see Table 1, also nucleotides 234-1166 of the nucleotide sequencepresent at GenBank accession number BX640939). Preferred polynucleotidesof the invention also include polynucleotides having a nucleotidesequence that is “substantially complementary” to a nucleotide sequencethat encodes a polypeptide according to the invention, or the complementof such nucleotide sequence. “Substantially complementary”polynucleotides can include at least one base pair mismatch, however thetwo polynucleotides will still have the capacity to hybridize. Forinstance, the middle nucleotide of each of the two DNA molecules5′-AGCAAATAT and 5′-ATATATGCT will not base pair, but these twopolynucleotides are nonetheless substantially complementary as definedherein. Two polynucleotides are substantially complementary if theyhybridize under hybridization conditions exemplified by 2×SSC(SSC: 150mM NaCl, 15 mM trisodium citrate, pH 7.6) at 55° C. Substantiallycomplementary polynucleotides for purposes of the present inventionpreferably share at least one region of at least 20 nucleotides inlength which shared region has at least 60% nucleotide identity,preferably at least 80% nucleotide identity, more preferably at least90% nucleotide identity, and most preferably at least 95% nucleotideidentity. Particularly preferred substantially complementarypolynucleotides share a plurality of such regions. Preferably thepolynucleotides have a nucleotide sequence that is at least 70%identical to SEQ ID NO:21. More preferably the polynucleotides have anucleotide sequence that is at least single unit percentages greaterthan 70% identical to SEQ ID NO:21, for example 71%, 72%, 73% identity,and so on to 100% identity to SEQ ID NO:21. Even more preferably, thepolynucleotides have a nucleotide sequence that is at least 80%identical, at least 90% identical, or at least 95% identity to SEQ IDNO:21. Most preferably, the polynucleotides have a nucleotide sequencethat is 100% identical to SEQ ID NO:21. A polynucleotide having at least70% identity to SEQ ID NO:21 has ER-α36 activity.

Percent identity between two polynucleotide sequences is generallydetermined by aligning the bases of the two polynucleotide sequences tooptimize the number of identical bases along the lengths of theirsequences; gaps in either or both sequences are permitted in making thealignment in order to optimize the number of identical bases, althoughthe bases in each sequence must nonetheless remain in their properorder. The two polynucleotide sequences are preferably compared usingthe Blastn program, version 2.0.11, of the BLAST 2 search algorithm,also as described by Tatusova et al. (NEMS Microbiol. Lett, 174, 247-250(1999)), and available on the world wide web athttp://www.ncbi.nlm.nih.gov/blast/b12seq/b12.html. Preferably, thedefault values for all BLAST 2 search parameters are used, includingreward for match=1, penalty for mismatch=−2, open gap penalty=5,extension gap penalty=2, gap x_dropoff=50, expect=10, wordsize=11, andoptionally, filter on. Locations and levels of nucleotide sequenceidentity between two polynucleotide sequences can also be readilydetermined using CLUSTALW multiple sequence alignment software (J.Thompson et al., Nucl. Acids Res., 22:4673-4680 (1994)), available atfrom the world wide web at www.ebi.ac.uk/clustalw/.

It should be understood that a polynucleotide that encodes a polypeptideof the invention is not limited to a polynucleotide that contains all ora portion of naturally occurring genomic or cDNA nucleotide sequence,but also includes the class of polynucleotides that encode suchpolypeptides as a result of the degeneracy of the genetic code. Forexample, the naturally occurring polynucleotide sequence SEQ ID NO:21 isbut one member of the class of nucleotide sequences that encodes apolypeptide having amino acid SEQ ID NO:20. The class of nucleotidesequences that encode a selected polypeptide sequence is large butfinite, and the nucleotide sequence of each member of the class can bereadily determined by one skilled in the art by reference to thestandard genetic code, wherein different nucleotide triplets (codons)are known to encode the same amino acid.

A polynucleotide that “encodes” a polypeptide of the inventionoptionally includes both coding and noncoding regions, and it shouldtherefore be understood that, unless expressly stated to the contrary, apolynucleotide that “encodes” a polypeptide is not structurally limitedto nucleotide sequences that encode a polypeptide but can include othernucleotide sequences outside (i.e., 5′ or 3′ to) the coding region. A“coding region” or “coding sequence” is a nucleotide sequence thatencodes a polypeptide and, when placed under the control of appropriateregulatory sequences expresses the encoded polypeptide. The boundariesof a coding region are generally determined by a translation start codonat its 5′ end and a translation stop codon at its 3′ end. An “exogenouscoding region” refers to a foreign coding region, i.e., a coding regionthat is not normally present in a cell, or a coding region that isnormally present in a cell but has been introduced into the cell byexperimental procedure, is operably linked to a regulatory region towhich it is not normally operably linked, or the combination thereof.

A polynucleotide of the invention can be linear or circular in topology.A polynucleotide can be, for example, a portion of a vector. A vectorcan provide for further cloning (amplification of the polynucleotide),i.e., a cloning vector, or for expression of the polypeptide encoded bythe coding region, i.e., an expression vector. A vector may include, butis not limited to, plasmid, phagemid, F-factor, virus, cosmid, or phage.The vector may be in a double-stranded or single-stranded linear orcircular form. The vector can also transform a prokaryotic or eukaryotichost either by integration into the cellular genome or existextrachromosomally (e.g., as an autonomous replicating plasmid with anorigin of replication). The polynucleotide in the vector can be underthe control of, and operably linked to, an appropriate promoter or otherregulatory sequence for transcription in vitro or in a host cell, suchas a eukaryotic cell, or a microbe, e.g. bacteria. Preferred examples ofeukaryotic cells include MDA-MB-231, Hela, CHO, and MCF10A cell lines. Aregulatory sequence, or regulatory region, refers to nucleotidesequences located upstream, within, or downstream of a coding sequence,and operably linked to, a coding sequence. Examples of regulatorysequences include enhancers, promoters, translation leader sequences,introns, and polyadenylation signal sequences. They include natural andsynthetic sequences as well as sequences that may be a combination ofsynthetic and natural sequences. Regulatory sequences are not limited topromoters. However, some suitable regulatory sequences useful in thepresent invention will include, but are not limited to, constitutivepromoters, tissue-specific promoters, development-specific promoters,inducible promoters and viral promoters. The term “operably linked”refers to a juxtaposition of components such that they are in arelationship permitting them to function in their intended manner. Aregulatory sequence is “operably linked” to a coding region when it isjoined in such a way that expression of the coding region is achievedunder conditions compatible with the regulatory sequence.

The vector may be a shuttle vector that functions in multiple hosts. Thevector may also be a cloning vector which typically contains one or asmall number of restriction endonuclease recognition sites at whichforeign DNA sequences can be inserted in a determinable fashion. Suchinsertion can occur without loss of essential biological function of thecloning vector. A cloning vector may also contain a marker gene that issuitable for use in the identification and selection of cellstransformed with the cloning vector. Examples of marker genes aretetracycline resistance or ampicillin resistance. Many cloning vectorsare commercially available (for instance Stratagene, New EnglandBiolabs, Clonetech). A vector may be an expression vector that containsregulatory sequences which direct the expression of a polynucleotidethat is inserted into the expression vector. Numerous vectors arecommercially available and are known in the art (Stratagene, La Jolla,Calif.; New England Biolabs, Beverly, Mass.). An expression vector maybe used in in vitro transcription and translation assays.

Methods to introduce a polynucleotide into a vector are well known inthe art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rdedition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)).Briefly, a vector into which a polynucleotide is to be inserted istreated with one or more restriction enzymes (restriction endonuclease)to produce a linearized vector having a blunt end, a “sticky” end with a5′ or a 3′ overhang, or any combination of the above. The vector mayalso be treated with a restriction enzyme and subsequently treated withanother modifying enzyme, such as a polymerase, an exonuclease, aphosphatase or a kinase, to create a linearized vector that hascharacteristics useful for ligation of a polynucleotide into the vector.The polynucleotide that is to be inserted into the vector is treatedwith one or more restriction enzymes to create a linearized segmenthaving a blunt end, a “sticky” end with a 5′ or a 3′ overhang, or anycombination of the above. The polynucleotide may also be treated with arestriction enzyme and subsequently treated with another DNA modifyingenzyme. Such DNA modifying enzymes include, but are not limited to,polymerase, exonuclease, phosphatase or a kinase, to create apolynucleotide that has characteristics useful for ligation of apolynucleotide into the vector.

The treated vector and polynucleotide are then ligated together to forma construct containing a polynucleotide according to methods known inthe art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rdedition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)).Briefly, the treated nucleic acid fragment and the treated vector arecombined in the presence of a suitable buffer and ligase. The mixture isthen incubated under appropriate conditions to allow the ligase toligate the nucleic acid fragment into the vector.

The invention further provides methods for making a polypeptide of theinvention and methods for making the polynucleotides that encode them.The methods include biological, enzymatic, and chemical methods, as wellas combinations thereof, and are well-known in the art. For example, apolynucleotide can be expressed in a host cell using standardrecombinant DNA technologies, it can be enzymatically synthesized invitro using a cell-free RNA based system, or it can be synthesized usingchemical technologies such as solid phase peptide synthesis. Whenrecombinant DNA technologies are used, the host cell can be, forexample, a bacterial cell, an insect cell, a yeast cell, or a mammaliancell.

The present invention also provides polynucleotides having promoteractivity. In one aspect, the promoter of the present invention includesa nucleotide sequence depicted at SEQ ID NO:22, or a portion thereof. Inanother aspect, a promoter of the present invention has a nucleotidesequence that is at least single unit percentages greater than 70%identical SEQ ID NO:22, for example 71%, 72%, 73% identity, and so on to100% identity to SEQ ID NO:22. Methods for determining percent identityare described herein. Even more preferably, the promoter has anucleotide sequence that is at least 80% identical, at least 90%identical, or at least 95% identity to SEQ ID NO:22. A promoter of thepresent invention has estrogen receptor (ER) regulated activity. As usedherein, ER regulated activity refers to increased expression of anoperably linked coding region in the presence of ER-α66, preferably, inthe presence of ER-α66 bound to an estrogen. A promoter of the presentinvention is expressed in an estrogen dependent and independent manner.A promoter of the present invention may be operably linked to a codingregion encoding a polypeptide, including a marker polypeptide. Examplesof marker polypeptides include detectable markers (for instance,fluorescent proteins, enzymes, antigenic markers, and the like) andselectable markers (polypeptides causing drug resistance, drugsusceptibility, or nutritional deficiency, or correcting a nutritionaldeficiency, and the like).

Antibodies

The invention provides antibodies that specifically bind to thepolypeptides and peptidomimetics of the invention. As used herein, anantibody that can “specifically bind” a polypeptide is an antibody thatinteracts only with the epitope of the antigen that induced thesynthesis of the antibody, or interacts with a structurally relatedepitope. An antibody that “specifically binds” to an epitope will, underthe appropriate conditions, interact with the epitope even in thepresence of a diversity of potential binding targets. In some aspects,an antibody of the present invention specifically binds to ER-α36 or aportion thereof, and does not specifically bind to ER-α66 or ER-α46.

Accordingly, the polypeptides and peptidomimetics of the invention andfragments thereof can be used as antigens to produce antibodies,including vertebrate antibodies, hybrid antibodies, chimeric antibodies,humanized antibodies, altered antibodies, univalent antibodies,monoclonal and polyclonal antibodies, Fab proteins, and single domainantibodies. For example, a polypeptide having SEQ ID NO:1 or a fragmentthereof, such as amino acids 13-27 of SEQ ID NO:1, can be used togenerate antibodies that specifically bind to ER-α36. A polypeptide orpeptidomimetic of the present invention, or fragments thereof, can bemodified by covalently linking them to an immunogenic carrier, such askeyhole limpet hemocyanin (KLH), bovine serum albumin, ovalbumin, mouseserum albumin, rabbit serum albumin, and the like.

If polyclonal antibodies are desired, a selected animal (e.g., mouse,rabbit, goat, horse or bird, such as chicken) may immunized with thedesired antigen. Serum from the immunized animal is collected andtreated according to known and routine methods. If serum containingpolyclonal antibodies to a polypeptide of the invention containsantibodies to other antigens, the polyclonal antibodies can be purifiedby immunoaffinity chromatography. Techniques for producing andprocessing polyclonal antisera are known in the art (see, for example,Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Pub.1988)).

Monoclonal antibodies directed against the polypeptides orpeptidomimetics of the present invention or fragments thereof can alsobe readily produced by one skilled in the art. The general methodologyfor making monoclonal antibodies by hybridomas is well known (see, forexample, Harlow et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Pub. 1988). Immortal antibody-producing cell lines (hybridimas)can be created by cell fusion, and also by other techniques such asdirect transformation of B lymphocytes with oncogenic DNA, ortransfection with Epstein-Barr virus. Panels of monoclonal antibodiesproduced against the polypeptides and peptidomimetics of the inventioncan be screened for various properties, for example epitope affinity.Other well known methods for making antibody include the use of phagedisplay techniques (see, for instance, Kay et al., Phage display ofpeptides and proteins: A laboratory manual. San Diego: Academic Press(1996))

An antibody of the invention may be derived from a “humanized”monoclonal antibody. Humanized monoclonal antibodies may be produced bytransferring mouse complementarity determining regions from heavy andlight variable chains of a mouse immunoglobulin into a human variabledomain, and then substituting human residues in the framework regions ofthe murine counterparts. The use of antibody components derived fromhumanized monoclonal antibodies obviates potential problems associatedwith the immunogenicity of murine constant regions. General techniquesfor cloning murine immunoglobulin variable domains are described(Orlandi et al., Proc. Nat'l Acad. Sci. USA, 86:3833 (1989), andtechniques for producing humanized monoclonal antibodies are described(Jones et al., Nature, 321:522 (1986); Riechmann et al., Nature, 332:323(1988)).

Antibody fragments of the invention can be prepared by routine knownmethods including proteolytic hydrolysis of the antibody or byexpression in E. coli of a polynucleotide encoding the fragment.Antibody fragments can be obtained by digestion (with, for instance,pepsin or papain) of whole antibodies by conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)2. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab′ fragments and an Fc fragment directly.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Antibodies can be screened to determine the identity of the epitope towhich they bind. An epitope refers to the site on an antigen, such as apolypeptide of the invention, to which the paratope of an antibodybinds. An epitope usually consists of chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andcan have specific three-dimensional structural characteristics, as wellas specific charge characteristics. Methods which can be used toidentify an epitope are known in the art (Harlow et al., Antibodies: ALaboratory Manual, page 319 (Cold Spring Harbor Pub. 1988).

Antibodies may be screened for their ability to specifically bind to apolypeptide or peptidomimetic of the present invention. For example,antibodies that specifically bind to the ER-α36 isoform or a portionthereof, but not the ER-α46 or α66 isoform, can be selected through useof methods routine in the art (see Kitajima et al., U.S. Pat. No.6,534,281, and Harlow et al., Antibodies: A Laboratory Manual, ColdSpring Harbor Pub. 1988).

The antibodies of the invention may be coupled to a large variety ofcompounds. Examples of compounds include detectable markers. Examples ofsuch detectable markers include fluorescent markers, enzymes,radioisotopes, and the like, such as avidin or biotin, which permit thedetection of an antibody. Methods to couple antibodies to detectablemarkers, and useful detectable markers, are known in the art. Suchantibodies are useful within automated systems for detection of ER-α36.An antibody can be covalently attached to a chemotherapeutic agent.Chemotherapeutic agents useful in the treatment of cancers such asbreast cancer and prostate cancer are known in the art. Examples ofchemotherapeutic agents include centchroman, delmadinone acetate,droloxifene, idoxifene, tamoxifen, raloxifene, toremifene, fulvestrantand faslodex, a bisphosphonate, calcitonin, tribolone, parathyroidhormone, or strontium ranelate. Other examples include a cytokine, or atoxin, such as diphtheria toxin A chain.

Compositions

The present invention also provides compositions including apolynucleotide, peptidomimetic, or antibody of the present invention.Such compositions typically include a pharmaceutically acceptablecarrier. As used herein “pharmaceutically acceptable carrier” includessaline, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Additional activecompounds can also be incorporated into the compositions.

The compositions of the invention may be prepared in many forms thatinclude tablets, hard or soft gelatin capsules, aqueous solutions,suspensions, and liposomes and other slow-release formulations, such asshaped polymeric gels. An oral dosage form may be formulated such thatthe polypeptide, peptidomimetic, or antibody is released into theintestine after passing through the stomach. Such formulations aredescribed in Hong et al., U.S. Pat. No. 6,306,434 and in the referencescontained therein.

Oral liquid compositions may be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid compositions may contain conventionaladditives such as suspending agents, emulsifying agents, non-aqueousvehicles (which may include edible oils), or preservatives.

A composition can be formulated for parenteral administration (e.g., byinjection, for example, bolus injection or continuous infusion) and maybe presented in unit dosage form in ampules, prefilled syringes, smallvolume infusion containers or multi-dose containers with an addedpreservative. The compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents.

Compositions suitable for rectal administration can be prepared as unitdose suppositories. Suitable carriers that may be included in thecomposition include those exemplified by saline solutions and othermaterials commonly used in the art.

For administration by inhalation, a composition can be convenientlydelivered from an insufflator, nebulizer or a pressurized pack or otherconvenient means of delivering an aerosol spray. Pressurized packs mayinclude a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, acomposition may take the form of a dry powder composition, for example,a powder mix of a modulator and a suitable powder base such as lactoseor starch. The powder composition may be presented in unit dosage formin, for example, capsules or cartridges or, e.g., gelatin or blisterpacks from which the powder may be administered with the aid of aninhalator or insufflator. For intra-nasal administration, a compositionmay be administered via a liquid spray, such as via a plastic bottleatomizer.

A composition can be formulated for transdermal administration. Acomposition can also be formulated as an aqueous solution, suspension ordispersion, an aqueous gel, a water-in-oil emulsion, or an oil-in-wateremulsion. A transdermal formulation may also be prepared byencapsulation of a composition within a polymer. The dosage form may beapplied directly to the skin as a lotion, cream, salve, or through useof a patch.

Compositions of the invention may also contain other ingredients such asflavorings, colorings, anti-microbial agents, and preservatives. Inaddition, a composition of the invention can include pharmaceuticallyactive ingredients, such as hormones, anti-necrotic agents,vasodilators, pharmaceutical agents and the like.

Toxicity and therapeutic efficacy of such compositions can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For a compound usedin the methods of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography. In oneaspect, the range of dosage for use in humans is an amount sufficient toresult in serum concentrations that are at least 10 micromolar (μM),preferably, at least 25 μM, more preferably, 50 μM.

The compositions can be administered one or more times per day to one ormore times per week, including once every other day. The skilled artisanwill appreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with an effective amount of acomposition can include a single treatment or, preferably, can include aseries of treatments.

Methods of Detection

The present invention provides methods for detecting a polypeptide ofthe present invention. The method typically includes providing a cell,analyzing the cell for a polypeptide of the present invention, anddetermining whether the cell expresses the polypeptide. The cell may beex vivo or in vivo. As used herein, the term “ex vivo” refers to a cellthat has been removed, for instance, isolated, from the body of asubject. Ex vivo cells include, for instance, primary cells (e.g., cellsthat have recently been removed from a subject and are capable oflimited growth or maintenance in tissue culture medium), and culturedcells (e.g., cells that are capable of extended growth or maintenance intissue culture medium). As used herein, the term “in vivo” refers to acell that is within the body of a subject. An in vivo cell may be a cellpresent in an organ or a tumor. The cell is preferably a mammalian cell,such as, for instance, mouse, rat, or primate (e.g., monkey, human),preferably, human. Preferred examples of cells include breast cells,such as a breast tumor cell, and prostate cells, such as a prostatetumor cell. A cell may be obtained from a subject by, for example,biopsy of human breast or prostate tissue. Samples obtained from nearlyany type of tissue may be used. Control cells may be cultured in vitroaccording to methods known in the art. Cells that do not express anER-α36 kDa estrogen receptor and thus can be used as a negative controlinclude HEK293 cells. Positive control cells include cells grown at lowcell density in the presence of serum, and BRCA1 negative cells.Preferably, cells are grown at low density in the presence of serum.Control cells may also be obtained from tissue samples through, forexample, biopsy.

In one aspect, the method includes analyzing the cell by contacting thecell with an antibody of the present invention. Whether a cell expressesa polypeptide of the present invention can be determined using detectionmethods that are routine and known in the art. Examples of immunoassaysinclude competitive and non-competitive assays such as radioimmunoassay,immunoenzymometric assay, immunofluorometric assay, orenzymoimmunoassays assays. Chemiluminescent methods with horseradishperoxidase, alkaline phosphatase, or other chemiluminescent detectionagents can also be used. Western blotting and chromatographic assays canalso be used within the method of the invention. An antibody of thepresent invention used to detect a polypeptide of the present inventionmay be coupled to a detectable marker and thereby detected directly, ora second antibody may be used. When detection methods are used thatpermit detection of the polypeptide in different areas of the cell, acell expressing an ER-α36 is typically considered ER-α36 positive whenthe polypeptide is associated predominantly with the plasma membrane andthe cytoplasm, and less than 20% of the signal is associated with thenucleus.

In another aspect, the method includes analyzing the cell by amplifyinga polynucleotide, preferably, an RNA polynucleotide (e.g., an mRNA), toform amplified polynucleotides. Preferably, a polynucleotide isamplified by polymerase chain reaction (PCR), preferably, by reversetranscriptase (RT) PCR. Methods for synthesizing a DNA polynucleotidefrom an RNA polynucleotide are known in the art and routine.Polynucleotides obtained from the cell are contacted with a primer pairthat will amplify a polynucleotide that includes SEQ ID NO:22 or SEQ IDNO:25, or the combination thereof. The presence of amplifiedpolynucleotides resulting from such a primer pair indicates the cellexpresses the estrogen receptor. As used herein, the term “primer pair”refers two oligonucleotides designed to flank a region of apolynucleotide to be amplified. One primer is complementary tonucleotides present on the sense strand at one end of a polynucleotideto be amplified and another primer is complementary to nucleotidespresent on the antisense strand at the other end of the polynucleotideto be amplified. The polynucleotide to be amplified can be referred toas the template polynucleotide. The nucleotides of a polynucleotide towhich a primer is complementary can be referred to as a target sequence.A primer can have at least about 15 nucleotides, preferably, at leastabout 20 nucleotides, most preferably, at least about 25 nucleotides.The conditions for amplifying a polynucleotide by PCR vary depending onthe nucleotide sequence of primers used, and methods for determiningsuch conditions are routine in the art.

After amplification, the presence of the amplified polynucleotides maybe determined, for instance, by gel electrophoresis. The amplifiedpolynucleotides can be visualized by staining (e.g., with ethidiumbromide) or labeling with a suitable label known to those skilled in theart, including radioactive and nonradioactive labels. Typicalradioactive labels include ³³P. Nonradioactive labels include, forexample, ligands such as biotin or digoxigenin as well as enzymes suchas phosphatase or peroxidases, or the various chemiluminescers such asluciferin, or fluorescent compounds like fluorescein and itsderivatives.

Optionally, the presence in a cell of an ER-α66 polypeptide, ER-α46polypeptide, ER-β, or the combination thereof, may also be determined.Methods for detecting the presence of ER-α66, ER-α46, ER43, or thecombination thereof, include the use of immunological detection usingantibody or polynucleotide based detection methods such as amplificationof a polynucleotide. When detection methods are used that permitdetection of the polypeptide in different areas of the cell, a cellexpressing an ER-α66 or ER-α46 is typically considered ER-α66 or ER-α46positive when the polypeptide is associated predominantly with thenucleus of the cell, e.g., greater than 90% of the signal is associatedwith the nucleus.

Approximately 70-80% of all breast cancers expresses ER-α66 and arereferred to as ER-positive breast cancer. These tumors usually grow moreslowly, are better differentiated, and are associated with a betteroverall prognosis (Clark, In: Harris J R, editor. Diseases of thebreast, volume 2. Lippincott Williams & Wilkins, 38:103-116 (2000)). Themethods for detecting the presence of a polypeptide of the presentinvention are useful as a diagnostic marker to differentiateestrogen-positive and estrogen-negative cancer, preferably, breastcancer. The results disclosed in the Examples herein strongly indicatethat estrogen signaling mediated by ER-α36 contributes to mammarytumorigenesis and suggest that ER-α36 may be involved in tumorigenesisof ER-α66 negative breast cancers. The results disclosed in the Examplesherein also indicate that a cell highly expressing a polypeptide of thepresent invention is more resistant to lower dose of an anti-estrogen,for instance, tamoxifen, than a cell that expresses high levels ofER-α66 but lower levels of ER-α36. Thus, methods for detecting thepresence of a polypeptide of the present invention permit theidentification of a new class of patients, i.e., ER-α66-negative andER-α36-positive. Methods for detecting the presence of a polypeptide ofthe present invention are also useful in determining the sensitivity ofa cell to an anti-estrogen, and providing information relevant to, forinstance, determining an appropriate course of treatment for anindividual. For instance, a physician may decide that a subject withER-α36-positive breast tumor cells may require higher doses of tamoxifento overcome resistance to lower levels.

Detection of the presence of ER-α66, ER-α46, ER-α36, ER-β, or thecombination thereof, also permits comparing the ratio of the estrogenreceptors. Determination of the ratio of two or more of the estrogenreceptors allows the sensitivity of a cell to an anti-estrogen, forinstance, tamoxifen, to be predicted. For instance, in this aspect ofthe invention the ratio of ER-α36 to ER-α46, the ratio of ER-α36 toER-α66, the ratio of ER-α36 to ER-13, or a combination thereof, in acell is determined and compared to the corresponding ratio in a controlcell. Such a ratio is referred to herein as an ER-α36 ratio. In oneexample, the control cell can be a cell that is refractory to treatmentwith an anti-estrogen. In another example, the control cell is a cellthat is not refractory to the anti-estrogen. If an above describedER-α36 ratio determined in the test cell is the same as the ER-α36 ratiodescribed in the control cell, then the test cell is classifiedaccording to the status of the control cell. For example, if the ER-α36to ER-α66 ratio of the test cell is the same as the ER-α36 to ER-α66ratio in a control cell known to be refractory to tamoxifen treatment,then the test cell is classified as being refractory to tamoxifentreatment. However, if the ER-α36 to ER-α66 ratio in the test cell isthe same as the ER-α36 to ER-α66 ratio in a control cell that is notrefractory to tamoxifen treatment, then the test cell is classified asnot being refractory to tamoxifen treatment. In another example, theER-α36 ratio determined in a test cell is compared to the correspondingratio in a control cell known to be refractory to tamoxifen treatment,and to the ratio in a control cell that is known not to be refractory totamoxifen treatment. The test cell is then classified as beingrefractory to tamoxifen treatment, or not refractory to tamoxifentreatment, as described above. An example of a control cell that isknown not to be refractory to tamoxifen treatment is MCF7 (ATTCCollection accession number HTB-22). An example of a control cell thatis known to be refractory to low dose tamoxifen treatment is MDA-MB-231.Breast cancer cells that are known to be refractory to tamoxifentreatment can also be used as control cells by comparing an ER-α36 ratioin a test cell.

Identification of Agents that Bind a Polypeptide of the PresentInvention

The present invention also provides methods for identifying an agentthat binds a polypeptide of the present invention. Such methods are alsoreferred to as screening assays. The method includes combining apolypeptide of the present invention with an agent, and determiningwhether the agent binds the polypeptide. Typically, determining whetheran agent binds the polypeptide includes detecting the formation of acomplex between the agent and the polypeptide. Methods for determiningthe complex include, for instance, directly detecting the binding of anagent to the polypeptide, and detecting the binding of the agent to thepolypeptide using a competition binding assay. The assay may be acell-free assay. The assay may be done in the presence or absence of anestrogen or anti-estrogen. Optionally, the method also includesdetermining whether the agent binds an ER-α66 polypeptide, such as apolypeptide with an amino acid sequence depicted at SEQ ID NO: 18.Preferably, an agent does not bind an ER-α66 polypeptide.

An agent can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including biologicallibraries, spatially addressable parallel solid phase or solution phaselibraries, synthetic library methods requiring deconvolution, the“one-bead one-compound” library method, and synthetic library methodsusing affinity chromatography selection. The biological library approachincludes peptide libraries, while the other four approaches areapplicable to peptide, nonpeptide oligomer, or small molecule librariesof compounds (Lam, AnticancerDrug Des. 12:145 (1997)). Examples ofmethods for the synthesis of molecular libraries can be found in the art(see, for example DeWitt et al. Proc. Natl. Acad. Sci. USA 90:6909(1993); Erb et al. Proc. Natl. Acad. Sci. USA 91:11422 (1994);Zuckermann et al. J. Med. Chem. 37:2678 (1994); Cho et al. Science261:1303 (1993); Carrell et at Angew. Chem. Int. Ed. Engl. 33:2059(1994); Carell et al. Angew. Chem. Int. Ed. Engl. 33:2061 (1994); andGallop et al. J. Med. Chem. 37:1233 (1994)). The sources for potentialagents to be screened include also include, for instance, fermentationmedia of bacteria and fungi, and cell extracts of plants and othervegetations.

Libraries of compounds maybe presented, for instance, in solution (e.g.Houghten Bio/Techniques 13:412-421 (1992)), or on beads (Lam Nature354:82-84 (1991)), chips (Fodor Nature 364:555-556 (1993)), bacteria(U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484;and 5,223,409), plasmids (Cull et al. Proc. Natl. Acad. Sci. USA89:1865-1869 (1992)), or phage (Scott et al. Science 249:386-390 (1990);Devlin Science 249:404-406 (1990); Cwirla et al. Proc. Natl. Acad. Sci.USA 87:6378-6382 (1990); and Felici J. Mol. Biol. 222:301-310 (1991)).

Determining the ability of an agent to bind to a polypeptide of thepresent invention can be accomplished, for example, by coupling theagent with a radioisotope or enzymatic label such that binding of theagent to the polypeptide of the present invention can be determined bydetecting the labeled compound in a complex. For example, agents can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemmission or byscintillation counting. Alternatively, agents can be enzymaticallylabeled with, for example, horseradish peroxidase, alkaline phosphatase,or luciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

In a similar manner, one may determine the ability of an agent to alter(e.g., stimulate or inhibit) the binding of a polypeptide of the presentinvention to a known ligand of the polypeptide, e.g., molecule withwhich a polypeptide of the present invention binds or interacts innature. An example of such a ligand is estrogen, as well as ananti-estrogen, such as tamoxifen. In a preferred aspect, the ability ofan agent to alter the binding of a polypeptide of the present inventionto bind to a ligand can be determined by monitoring the activity of thepolypeptide of the invention.

In yet another aspect, an assay of the present invention includescontacting a polypeptide of the present invention with an agent anddetermining the ability of the agent to bind to the polypeptide. Bindingof the agent to the polypeptide can be determined either directly orindirectly as described above. In a preferred aspect, the assay includescontacting a polypeptide of the present invention with a ligand known tobind a polypeptide of the present invention to form an assay mixture,contacting the assay mixture with an agent, and determining the abilityof the agent to preferentially bind to the polypeptide as compared tothe ligand.

In another aspect, an assay includes contacting a polypeptide of thepresent invention with an agent and determining the ability of the agentto alter (e.g., stimulate or inhibit) the activity of the polypeptide ofthe present invention.

In a further aspect, an assay includes screening for agents that alter(e.g., stimulate or inhibit) the ability of a polypeptide of the presentinvention to regulate transcriptional transactivation of an estrogenresponse element, including, for instance, activities mediated by theAF-1 and/or AF-2 domains of ER-α66. Preferably, a fusion polypeptideincluding a polypeptide of the present invention and a polypeptidehaving a transcriptional activation domain, or a transcriptionalrepressor, domain, is used. An example of a polypeptide having atranscriptional activation domain is VP-16, and other usefulpolypeptides having a transcriptional activation domain or atranscriptional repressor domain are known in the art. Typically, such afusion polypeptide is used in conjunction with a polynucleotide havingan estrogen response element upstream of a promoter and an operablylinked coding sequence. A variety of promoters can be used, including,for instance, a thymidine kinase promoter. Preferably, the operablylinked coding region encodes a detectable marker, such as luciferase, ora fluorescent polypeptide such as green fluorescent protein. In oneaspect, the fusion polypeptide with transcriptional activation domain,polynucleotide, and agent are combined under conditions that promoteexpression of the coding region present on the polynucleotide in theabsence of the agent, and the effect of the agent in alteringtranscription is determined. Optionally, an ER-α66 polypeptide and/or anERβ polypeptide may also be present, and the assay used to identifyagents that alter (e.g., stimulate or inhibit) the ability of apolypeptide of the present invention to modulate ligand-dependent andligand-independent transcriptional activities of ER-α66 polypeptide orERβ. Preferably, both the fusion polynucleotide and the polynucleotidethat includes an estrogen response element upstream of a promoter and anoperably linked coding sequence are present in a cell. Without intendingto be limiting, it is expected that agents that alter the ability of apolypeptide of the present invention to regulate transcriptionaltransactivation may include agents that alter the conformation of apolypeptide of the present invention to increase or decrease the abilityof the

In the assays, it may be desirable to immobilize either a polypeptide ofthe present invention, its ligand, or the agent to facilitate separationof complexed from uncomplexed forms of one or both of the molecules, aswell as to accommodate automation of the assay. In one embodiment, afusion protein can be provided that adds a domain that allows thepolypeptide of the present invention to be bound to a matrix. Forexample, fusion polypeptides with glutathione-S-transferase can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione-derivatized microtitre plates, which are thencombined with the agent, and the mixture incubated under conditionsconducive to complex formation (e.g. at physiological conditions forsalt and pH). Following incubation, the beads or microtitre plate wellscan be washed to remove any unbound components and complex formation ismeasured either directly or indirectly, for example, as described above.Alternatively, the complexes can be dissociated from the matrix, and thelevel of binding determined.

Other techniques for immobilizing polypeptides on matrices can also beused in the screening assays of the invention. For example, apolypeptide of the present invention can be immobilized usingconjugation of biotin and streptavidin. A polypeptide of the presentinvention can be biotinylated using biotin-NHS (N-hydroxy-succinimide)with techniques well known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and, for instance, immobilized in the wellsof streptavidin-coated 96-well plates (Pierce Chemicals). Alternatively,antibodies reactive with a polypeptide of the present invention can bederivatized to the wells of the plate, and unbound polypeptide of thepresent invention trapped in the wells by antibody conjugation. Methodsfor detecting such complexes, in addition to those described above forthe GST-immobilized complexes, include immunodetection of complexesusing antibodies reactive with antibody that specifically binds apolypeptide of the present invention.

The present invention also includes novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

Methods of Treatment

The present invention is further directed to methods for treatingcertain diseases in a subject. The subject is a mammal, preferably ahuman. As used herein, the term “disease” refers to any deviation fromor interruption of the normal structure or function of a part, organ, orsystem, or combination thereof, of a subject that is manifested by acharacteristic symptom or set of symptoms. Diseases include cancersdependent upon signalling via steroid hormone receptors, such asestrogen receptors. Examples of such diseases are referred to asestrogen-related cancers and include breast cancer and prostate cancer.Typically, whether a subject has a disease, and whether a subject isresponding to treatment, is determined by evaluation of symptomsassociated with the disease. As used herein, the term “symptom” refersto objective evidence of a disease present in a subject. Symptomsassociated with diseases referred to herein and the evaluation of suchsymptoms are routine and known in the art. Examples of symptoms ofcancers dependent upon signalling via steroid hormone receptors include,for instance, the presence and size of tumors, and the presence andamount of biomarkers. Biomarkers are compounds, typically polypeptides,present in a subject and indicative of the progression of cancer.Examples of biomarkers include, for instance, Her-2 expression, andcyclin D1 expression.

Treatment of a disease can be prophylactic or, alternatively, can beinitiated after the development of a disease. Treatment that isprophylactic, for instance, initiated before a subject manifestssymptoms of a disease, is referred to herein as treatment of a subjectthat is “at risk” of developing a disease. An example of a subject thatis at risk of developing a disease is a person having a risk factor,such as a genetic marker, that is associated with the disease. Examplesof genetic markers indicating a subject has a predisposition to developcertain cancers such as breast, or prostate cancer include alterationsin the BRAC1 and/or BRAC2 genes. Treatment can be performed before,during, or after the occurrence of the diseases described herein.Treatment initiated after the development of a disease may result indecreasing the severity of the symptoms of one of the conditions, orcompletely removing the symptoms.

In some aspects, the methods typically include contacting a cell with acomposition including an effective amount of an agent that inhibits theactivity of a polypeptide of the present invention, for instance, anagent identified using a method described herein. Preferably, such anagent binds to a polypeptide of the present invention. In some aspects,the agent is preferably not an anti-estrogen. As used herein, an“effective amount” is an amount effective to inhibit in a cell theactivity of a polypeptide of the present invention, decrease symptomsassociated with a disease, or the combination thereof. In one aspect, acomposition may include an effective amount of an antibody of thepresent invention. Preferably, an antibody is covalently attached to achemotherapeutic agent, such as, for instance, tamoxifen. Thecomposition may optionally include other chemotherapeutic agents.Whether an agent or antibody, preferably, antibody, is expected tofunction in this aspect of the invention can be evaluated using ex vivomodels and animal models. Such models are known in the art and aregenerally accepted as representative of disease or methods of treatinghumans. A preferred example of such an animal model is the nude mouse.For instance, breast cancer cells can be inoculated into the mammaryfatpad of ovariectomized female nude mice, and lesion formation followedand evaluated, for example, by palpation, measurement by verniercalipers, and tumor weight. Transgenic animal models are also available.For instance, models for the study of prostate cancer such as the TRAMPmodel (see, for instance, Greenberg et al., Proc. Natl. Acad. Sci. USA,92:2429-3443 (1995)) and for breast cancer such as the MMTV-Wnt-1 model(see, for instance, Tsukamoto et al., Cell, 55:619-625 (1988)) arecommonly accepted as models for human disease.

In another aspect, a cell is contacted with a composition including apolynucleotide, where the polynucleotide causes the post-transcriptionalsilencing of a coding region encoding a polypeptide of the presentinvention. Such a polynucleotide is referred to herein as a silencingpolynucleotide. The silencing polynucleotide may be introduced into acell as an RNA polynucleotide, or as a vector including a DNApolynucleotide that encodes and will express the RNA polynucleotide.More than one type of polynucleotide can be administered. For instance,two or more polynucleotides that are designed to silence the same mRNAcan be combined and used in the methods herein. Alternatively, two ormore polynucleotides can be used together where the polynucleotides areeach designed to silence different mRNAs.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides, deoxynucleotides, ora combination thereof, and includes both single-stranded molecules anddouble-stranded duplexes. A polynucleotide can be obtained directly froma natural source, or can be prepared with the aid of recombinant,enzymatic, or chemical techniques. Preferably, a polynucleotide of thepresent invention is isolated. As used herein, a “target coding region”and “target coding sequence” refer to the coding region whose expressionis inhibited by a silencing polynucleotide. As used herein, a “targetmRNA” is an mRNA encoded by a target coding region. An example of atarget coding region is the nucleotide sequence encoding an ER-α36 (SEQID NO: 21), the 5′ flanking nucleotide sequence (SEQ ID NO:20), or the3′ flanking nucleotide sequence, including the nucleotide sequenceencoded by exon 9 (SEQ ID NO:25).

Silencing polynucleotides include double stranded RNA (dsRNA)polynucleotides. The sequence of a silencing polynucleotide includes onestrand, referred to herein as the sense strand, of between 16 to 30nucleotides, for instance, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides. The sense strand is substantiallyidentical, preferably, identical, to a target mRNA. As used herein, theterm “identical” means the nucleotide sequence of the sense strand hasthe same nucleotide sequence as a portion of the target mRNA. As usedherein, the term “substantially identical” means the sequence of thesense strand differs from the sequence of a target mRNA at 1, 2, or 3nucleotides, preferably 1 nucleotide, and the remaining nucleotides areidentical to the sequence of the mRNA. These 1, 2, or 3 nucleotides ofthe sense strand are referred to as non-complementary nucleotides. Whena silencing polynucleotide includes a sense strand that is substantiallyidentical to a target mRNA, the 1, 2, or 3 non-complementary nucleotidesare preferably located in the middle of the sense strand. For instance,if the sense strand is 21 nucleotides in length, the non-complementarynucleotides are typically at nucleotides 9, 10, 11, or 12, preferablynucleotides 10 or 11. The other strand of a dsRNA polynucleotide,referred to herein as the anti-sense strand, is complementary to thesense strand. The term “complementary” refers to the ability of twosingle stranded polynucleotides to base pair with each other, where anadenine on one polynucleotide will base pair to a thymine or uracil on asecond polynucleotide and a cytosine on one polynucleotide will basepair to a guanine on a second polynucleotide. The silencingpolynucleotides also include the double stranded DNA polynucleotidesthat correspond to the dsRNA polynucleotides. Also included are thesingle stranded RNA polynucleotides and single stranded DNApolynucleotides corresponding to the sense strands and anti-sensestrands disclosed herein. It should be understood that the sequencesdisclosed herein as DNA sequences can be converted from a DNA sequenceto an RNA sequence by replacing each thymidine nucleotide with a uracilnucleotide. Without intending to be limiting, the polynucleotides of thepresent invention cause the post-transcriptional silencing of a targetcoding region. Modifications to polynucleotides for use in silencing areknown in the art and the silencing polynucleotides can be so modified.

The sense and anti-sense strands of a dsRNA silencing polynucleotide ofthe present invention may also be covalently attached, typically by aspacer made up of nucleotides. Such a polynucleotide is often referredto in the art as a short hairpin RNA (shRNA). Upon base pairing of thesense and anti-sense strands, the spacer region forms a loop. The numberof nucleotides making up the loop can vary, and loops between 3 and 23nucleotides have been reported (Sui et al., Proc. Natl. Acad. Sci. USA,99, 5515-5520 (2002), and Jacque et al., Nature, 418, 435-438 (2002)).

A silencing polynucleotide causes the post-transcriptional inhibition ofexpression, also referred to as silencing, of a target coding region.Without intending to be limited by theory, after introduction into acell a silencing polynucleotide will hybridize with a target mRNA andsignal cellular endonucleases to cleave the target mRNA. The result isthe inhibition of expression of the polypeptide encoded by the mRNA.Whether the expression of a target coding region is inhibited can bedetermined by, for instance, measuring a decrease in the amount of thetarget mRNA in the cell, measuring a decrease in the amount ofpolypeptide encoded by the mRNA, or by measuring a decrease in theactivity of the polypeptide encoded by the mRNA. A silencingpolynucleotide can be present in a vector. A silencing polynucleotidecan be present in a vector as two separate complementarypolynucleotides, each of which can be expressed to yield a sense and anantisense strand of the dsRNA, or as a single polynucleotide containinga sense strand, a loop region, and an anti-sense strand, which can beexpressed to yield an RNA polynucleotide having a sense and an antisensestrand of the dsRNA.

A silencing polynucleotide can be designed using methods that areroutine and known in the art. For instance, a silencing polynucleotidemay be identified by scanning the coding region AA dinucleotidesequences; each AA and the downstream (3′) consecutive 16 to 30nucleotides of the mRNA can be used as the sense strand of a candidatepolynucleotide. A candidate polynucleotide is the polynucleotide that isbeing tested to determine if it decreases expression of one of apolypeptide of the present invention. The candidate polynucleotide canbe identical to nucleotides located in the region encoding thepolypeptide, or located in the 5′ or 3′ untranslated regions of themRNA. Optionally and preferably, a candidate polynucleotide is modifiedto include 1, 2, or 3, preferably 1, non-complementary nucleotides.Other methods are known in the art and used routinely for designing andselecting candidate polynucleotides. A silencing polynucleotide may, butneed not, begin with the dinucleotide AA at the 5′ end of the sensestrand. A candidate polynucleotide may also include overhangs of 1, 2,or 3 nucleotides, typically on the 3′ end of the sense strand, theanti-sense strand, or both. Candidate polynucleotides are typicallyscreened using publicly available algorithms (e.g., BLAST) to comparethe candidate polynucleotide sequences with coding sequences. Those thatare likely to form a duplex with an mRNA expressed by a non-targetcoding region are typically eliminated from further consideration. Theremaining candidate polynucleotides may then be tested to determine ifthey inhibit expression of one of the polypeptides described herein.

In general, candidate polynucleotides are individually tested byintroducing a candidate polynucleotide into a cell that expressespolypeptide of the present invention. The candidate polynucleotides maybe prepared in vitro and then introduced into a cell. Methods for invitro synthesis include, for instance, chemical synthesis with aconventional DNA/RNA synthesizer. Commercial suppliers of syntheticpolynucleotides and reagents for such synthesis are well known. Methodsfor in vitro synthesis also include, for instance, in vitrotranscription using a circular or linear vector in a cell free system.

The candidate polynucleotides may also be prepared by introducing into acell a construct that encodes the candidate polynucleotide. Suchconstructs are known in the art and include, for example, a vectorencoding and expressing a sense strand and an anti-sense strand of acandidate polynucleotide, and RNA expression cassettes that include thesequence encoding the sense strand and an anti-sense strand of acandidate polynucleotide flanked by operably linked regulatorysequences, such as an RNA polymerase III promoter and an RNA polymeraseIII terminator, that result in the production of an RNA polynucleotide.The cell can be ex vivo or in vivo. Candidate polynucleotides may alsobe tested in animal models.

When evaluating whether a candidate polynucleotide functions to inhibitexpression of one of the polypeptides described herein, the amount oftarget mRNA in a cell containing a candidate polynucleotide can bemeasured and compared to the same type of cell that does not contain thecandidate polynucleotide. Methods for measuring mRNA levels in a cellare known in the art and routine. Such methods include quantitativeRT-PCR. Primers and specific conditions for amplification of an mRNAvary depending upon the mRNA, and can be readily determined by theskilled person. Other methods include, for instance, Northern blotting,and array analysis.

Other methods for evaluating whether a candidate polynucleotidefunctions to inhibit expression of one of the polypeptides describedherein include monitoring the polypeptide. For instance, assays can beused to measure a decrease in the amount of polypeptide encoded by themRNA, or to measure a decrease in the activity of the polypeptideencoded by the mRNA. Methods for measuring a decrease in the amount of apolypeptide include assaying for the polypeptide present in cellscontaining a candidate polynucleotide and comparing to the same type ofcell that does not contain the candidate polynucleotide. For instance,an antibody of the present invention may be used in, for example,Western immunoblot, immunoprecipitation, or immunohistochemistry.Antibodies to each of the polypeptides described herein are commerciallyavailable. Methods for measuring a decrease in the activity of one of apolypeptide of the present invention may also be used.

Kits

The invention provides kits that contain reagents that can be used inthe methods of the present invention, including, for instance,determining if a cell expresses a polypeptide of the present invention.Such kits can contain packaging material and an antibody of the presentinvention. Such kits may also be used by medical personal for theformulation of compositions, such as pharmaceutical compositions, thatcontain an antibody of the invention.

The packaging material provides a protected environment for theantibody. For example, the packaging material may keep the antibody frombeing contaminated. In addition, the packaging material may keep anantibody in solution from becoming dry. Examples of suitable materialsthat can be used for packaging materials include glass, plastic, metal,and the like. Such materials may be silanized to avoid adhesion of anantibody to the packaging material.

In one example, the invention provides a kit that includes packagingmaterial, a first antibody that specifically binds to a polypeptide ofthe invention, and a second antibody that specifically binds to anER-α66 polypeptide. The kit may optionally include additional componentssuch as buffers, reaction vessels, secondary antibodies, and syringes.

EXAMPLES Example 1 Caveolin-1 Haploinsufficiency Produces Activation ofER-α Expression and Estrogen Stimulated Transformation of Normal BreastEpithelial Cells

A gene-trapped library of cell clones from normal human mammaryepithelial MCF10A cells was prepared through use of a poly-A trapretrovirus vector (RET) obtained from Dr. Philip Leder's laboratory atHarvard Medical School (Ishida et. al., Nucl. Acid Res., 27:580 (1999)).Briefly, this vector used an improved poly-A trap strategy for theefficient identification of functional genes regardless of theirexpression status in target cells. A combination of a strong spliceacceptor and an effective polyadenylation signal assures the completedisruption of the function of “trapped” genes. Inclusion of apromoterless GFP cDNA in the RET vector allows the expression pattern ofthe trapped gene to be easily monitored in living cells. A retroviruscontaining the RET vector was used to infect MCF10A cells. The cellswere then screened for G418-resistant to establish a gene-trappedlibrary of MCF10A cells. After selection by G418 for three weeks, GFPexpression, under the control of the endogenous promoter of the“trapped” gene, was monitored and G418 resistant and GFP expressingclones were then selected. This library represented 3×10⁵ independentinfected clones in which one allele of a functional gene was disruptedby the RET vector.

It was thought that loss of expression of genes with tumor suppressionactivity could confer the transformed phenotype to normal MCF10A cells.A soft-agar cloning assay was performed and cells from the gene-trappedcell library that acquired anchorage-independent growth, acharacteristic of the transformed phenotype, were identified. More than100 positive colonies (>30 cells) from the library of G418-resistantcells grew in soft-agar while the parental MCF10A cells did not. Twentycell clones were isolated, expanded, and then selected again insoft-agar containing regular serum plus 10-8 M 17b-estradiol (E2) anddextrin coated charcoal-stripped serum that lacks steroid hormones forthree weeks. Four cell clones (ST1, ST3, ST4 and ST6) that exhibitedaccelerated growth in soft-agar containing extra E2 were isolated andexpanded (FIG. 3).

3′-RACE, which permits the capture of unknown 3′ mRNA sequences that liebetween the exon of a candidate gene and the poly-A tail, was used toclone potential genes whose disruption leads to MCF10A celltransformation. Transformation of the MCF10A cells was thought to be dueto positive estrogen signaling. The purified PCR fragments resultingfrom the RACE procedure were cloned and sequenced. Using a BLASTNsearch, the DNA sequences from two clones (ST1 and ST3) were matchedidentically to the sequence of caveolin-1 (Cav-1) exon-3 located onchromosome 7 (GenBank accession number XM048940). This result indicatedthat an allele of the Cav-1 gene was disrupted in at least 2 clones. Inaddition to Cav-1, two other genes were identified using the sametechnique. The gene disrupted in clone ST4 was SPRR1B (GenBank accessionnumber NT-004441.5), a member of the cornifin/small proline-rich proteinfamily involved in structural organization of cornified cell envelopes.Another gene from clone ST6 is a putative novel gene (GenBank accessionnumber 6599139) with no known function.

Cav-1 protein levels were analyzed in parental MCF10A cells to testwhether expression levels of Cav-1 were decreased in the cav-1 genetrapped cells. Four cell clones (ST1, 3, 4 and 6) described above, andMCF10A-Ha-ras, (MCF10A cells transformed by a Ha-ras mutant) wereanalyzed. Compared to the levels in parental MCF10A cells, Cav-1 proteinlevels were about 2-fold lower in all transformed cells as demonstratedby Western blot analysis (FIG. 4). This data is consistent with Cav-1expression being decreased when only one functional allele of the cav-1gene is functional in ST1 and ST3 cells. This indicates that Cav-1haploinsufficiency created by “gene trapping” leads to transformationstimulated by E2. This data also indicates that downregulation of Cav-1is also involved in transformation resulted from the disruption of othergenes in ST4 and ST6 cells.

To determine the mechanism by which Cav-1 haploinsufficiency leads toestrogen-stimulated cell growth and transformation, the expressionlevels of ER-α and ER-β in the transformed cells described above wasexamined. It was found that all of the four transformed cell clonesexpressed ER-α at a level comparable to that in the Ha-ras transformedcells, whereas parental MCF10A cells and HBL-100 cells, another normalmammary epithelial cell line, expressed undetectable levels of ER-α(FIG. 5). ER-β expression was without any change in all of the cellstested (FIG. 5). This data indicated that ER-α expression was activatedand that estrogen signaling in these transformed cells is responsiblefor the estrogen-stimulated cell growth on soft-agar. It has been shownbefore that both ER-α expression and estrogen signaling are activated inHa-ras transformed cells (Shekhar et. al., Int. J. Oncol., 13:907 (1998)and Shekhar et. al., Am. J. of Pathl., 152:1129 (1998)). The presentresults indicate that the Ras/MAPK pathway is involved in the regulationof ER-α expression and positive estrogen signaling.

Activation of the MAPK pathway in these transformed cells was analyzedby examining the phosphorylation levels of ERK1/2 using phospho-specificantibodies. It was found that ERK1/2 are highly and constitutivelyphosphorylated in all transformed cells but not in MCF10A cells (FIG.6).

Taken together, these data indicate that the Cav-1/Ras/MAPK pathway isinvolved in the activation of ER-α expression during human breast cancerdevelopment and cooperates with estrogen signaling pathway to stimulatetransformed cell proliferation.

Example 2 Identification, Cloning, Expression and Characterization of anIsoform of Estrogen Receptor Alpha (ER-α36)

During the course of the work described above, three protein bands(66-kDa, 46-kDa and 36-kDa) were consistently observed in western blotanalysis using the Rat anti-ER-α antibody (clone H222) from ResearchDiagnostic, INC. The H222 antibody recognizes the ligand-binding domainof ER-α. To exclude the possibility that 46-kDa and 36-kDa protein bandswere the degradation products of ER-α66, as suggested by a previousreport (Abbondanza et. al., Steroids, 58:4 (1993)), cells were lysed inculture plates using a buffer containing 8 M urea and tested by westernblot analysis. Three distinct bands were readily observed in Cav-1haploinsufficient cells, ST1 and ST3, and MCF7 breast cancer cells (FIG.7). These results indicated the existence of ER-α isoforms that share asimilar epitope that is recognized by the antibody H222.

Through a literature search, it was found that a 46-kDa isoform of ER-αhad been cloned that functions as a dominant-negative inhibitor oftransactivation mediated by the AF-1 domain of ER-α66 (Flouriot et. al.,EMBO J., 19:4688 (2000)). A continued search identified a clone from anormal human edometrium cDNA library (RZPD clone number: DKFZp686N23123)that contains a 5.4 kb cDNA. This cDNA clone harbors a 310 amino acidopen reading frame that theoretically can produce a protein with apredicted molecular weight of 35.7 kDa. The cDNA sequence of theopen-reading frame matched 100% to DNA sequence of the exons 2 to 6 ofthe ER-α66 gene. The 5′ untranslated region (5′UTR) of the cDNA showed100% homology to the DNA sequence of the first intron of the ER-α66 genefrom 734 to 907 (the first base pair of the 34,233 by first intron ofER-α66 gene was designated as 1). Thus, it was determined that thetranscript of ER-α36 is initiated from a previously unidentifiedpromoter in the first intron of the ER-α66 gene.

A small, non-coding novel exon from 734 to 907 of the first intron ofthe ER-α66 gene was designated as “exon 1′”. The exon 1′ is then spliceddirectly into the exon 2 of the ER-α66 gene and continues from exon 2 toexon 6 of the ER-α66 gene. Exon 6 is then spliced to an exon located64,141 by downstream of the ER-α66 gene (GeneBank accession numberAY425004, see Table 1). The cDNA sequence encoding the last 27 aminoacids and the 4,293 by 3′ untranslated region was matched 100% to acontinuous sequence from the genomic sequence of clone RP1-1304 onchromosome 6q24.2-25.3 (GeneBank accession number AL078582), indicatingthe remaining cDNA sequence of this novel ER-α isoform is transcribedfrom one exon of 4,374 by located downstream of the previously reportedER-α66 gene. This exon is thus designated as exon 9 to reflect the extraexon beyond the previous reported eight exons (FIG. 8). All of thesesplicing events are supported by the identification of perfect splicedonors and acceptors at the splice juncture. The protein ER-α36 can beproduced from a perfect Kozak sequence located in the second exon, thesame initiation codon used to produce ER-α46 (Flouriot et. al., EMBO J.,19:4688 (2000)). ER-α36 differs from ER-α66 by lacking bothtranscriptional activation domains, AF-1 and AF-2, but retaining thedimerization, DNA-binding and partial ligand-binding domains. It alsopossesses an extra, unique 27 amino acid domain to replace the last 138amino acids encoded by exon 7 and 8 of the ER-α66 (FIG. 1). Here, thisnovel isoform of ER-α is herein named ER-α36.

The open reading frame encoding ER-α36 was obtained by using the PCRfrom the Marathon Ready cDNA prepared from human placenta RNA(Clonetech) according to the procedure described by the manufacture. ThePCR primer pairs are designed according to the cDNA sequence of686N23123. The 5′ primer is 5′-CGGAATTCCGAAGGGAAGTATGGCTATGGAATCC-3′(SEQ ID NO:23) with an EcoRI site at the end, and the 3′ primer is5′-CGGGATCCAGAGGCTTTAGACACGAGGAAAC-3′ (SEQ ID NO:24) with a BamHI siteat the end. The PCR product was subjected to electrophoresis on a 1%agarose gel, and an expected 1.1 kb DNA fragment was observed (FIG. 9).The DNA fragment was purified, digested with EcoRI and BamHI, clonedinto a pBluescript vector (pBS-ER-α36) and fully sequenced. The sequenceshowed 100% identity to the cDNA clone DKFZp686N23123, indicated thatER-α36 is a naturally occurring isoform of ER-α, that can be cloned fromanother source. The predicted amino acid sequence encoded by theopen-reading frame is shown in FIG. 10.

Transient transfection assays were performed in human embryonic kidney293 cells using expression vectors containing ER-α66, ER-α46 and ER-α36to test whether the cloned cDNA will produce the ER-α36 protein. Wholecell extracts from these transfected cells and MCF7 cells were subjectedto western blot analysis with the monoclonal antibody H222 raisedagainst the ligand-binding domain of ER-α (Abbondanza et. al., Steroids,58:4 (1993)). A 36 kDa protein that was recognized by the H222 antibodywas produced in ER-α36 vector transfected cells (FIG. 11). The size ofthis protein and its failure to react with the antibody H226 directed tothe B-domain of the ER-α66, and with the antibody HC 20 which recognizesthe C-terminal of ER-α66, indicates that the ER-α isoform lacked boththe N-terminus and C-terminus of ER-α66, resulting in an ER-α lackingboth AF-1 and AF-2 domains.

A series of computer searches were performed on the ER-α36 protein.FindMod and SCANPROSITE algorithms predicted three myristoylation sitesin ER-α36, suggesting that it may localize in the peripheral membrane.This is in agreement with the k-nearest neighbors (PSPORT II) algorithmthat predicts 21.7%, 34.8%, 17.4%, and 26% of ER-α36 is localized to thenucleus, cytoplasm, mitochondria, and membrane fractions, respectively.This is similar to the prediction for ER-α46 (26.1%, 30.4%, 17.4%, and26.1%, respectively). By contrast, 73.9% 8.7%, 0.1% and 17.3% of ER-α66carries comparative predictions. Thus, the differentialcompartmentalization of ER-α66, ER-α46, and ER-α36 indicates that thefunctional site and primary role of each receptor may be different.

A computer search was also performed on the putative 5′ flanking regionof the gene encoding ER-α36 that is located in the first intron ofER-α66 gene. A TATA binding protein (TBP) recognition sequence was foundupstream of the cDNA start site and several Sp1, NF-κB and Ap1 bindingsites in the 5′ flanking region (FIG. 12). A perfect half estrogenresponse element (ERE) site was identified at the 5′ upstream region ofER-α36, indicating that ER-α36 is subjected to E2-mediatedtranscriptional regulation.

Example 3 ER-α36 Mediates Membrane-Initiated Estrogen Signaling and isExpressed in ER-Negative Breast Cancer Methods

Cell culture, establishment of stable cell lines and membrane-labelingwith E2-BSA-FITC. MCF10A cells were obtained from Karmanos CancerInstitute at Detroit, Mich., and human embryonic kidney 293 cells, andall breast cancer cells were obtained from ATCC. All cells weremaintained at 37° C. in a 5% CO₂ atmosphere in appropriate tissueculture medium. To establish stable cells that express recombinantER-α36, HEK293 cells were plated at a density of 1×10⁵ cells per 60-mmdish and transfected 24 hours later with ER-α36 expression vector drivenby the cytomagalovirus (CMV) promoter using the FuGene6 transfectionreagent (Roche Molecular Biochemicals). The ER-α36 expression vector wasconstructed by cloning a 1.1-kb EcoRI-BamHI cDNA fragment of ER-α36 frompBS-ER-α36 into the EcoRI and BamHI sites of mammalian expression vectorpCB6+. Empty vector was also transfected into cells to serve ascontrols. Forty-eight hours after transfection, the cells were replatedand selected with 500 μg/ml of G418 (Invitrogen) for two weeks. Theresulting uncloned population of G418-resistant cells was expanded togenerate cells used for further analysis. To label the cell surface ofstable cells that express recombinant ER-α36, cells were labeled at 4°C. for 15 minutes with 1 μM fluorescein isothiocyanate (FITC)-labeledBSA covalently attached to E2β-hemisuccinate (Sigma), fixed in freshlyprepared 4% paraformaldehyde and mounted with mounting solutioncontaining DAPI for microscopic evaluation.

Cellular stimulation with estrogens and anti-estrogens, and MTT assay.Before treatment, cells were cultured in phenol-red free medium with2.5% dextrin-coated charcoal-stripped Fetal Calf Serum for 48-72 hours,and then washed with PBS and placed in fresh phenol red-free, serum-freemedium containing 0.1 μg/ml of BSA and 5 μg/ml of insulin for 12 hours.Stimulation of quiescent cells was carried out at 37° C. in serum freemedium for different period of time. Different estrogens andantiestrogens were purchased from Steraloids Inc. BSA-E2β was obtainedfrom Sigma.

For 3-(4,5-dimethylthiazo)-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assays, cells in suspension were added to each well of a 96-well cultureplate for a final concentration of 1×10³ cells/well, and incubated for24 hours at 37° C. in a CO₂ incubator. Media containing 10 nM E2P, 10 nMof Tamoxifen or 4OH-Tamoxifen or 7.2 nM UO126 (Calbiochem) were added toeach well for 48 hours. MTT assay was performed with the CellTiter96Aqueous One Solution Cell Proliferation Assay Kit (Bio Rad) as themanufacture recommended. A microplate reader (Promega) was used tomeasure absorbance at 490 nm.

Cell fractionation assay. Cell fractionation was done as described byMarquez et al. (Oncogene, 20, 5420-5430 (2001)).

Western blot analysis, indirect immunofluorescence and antibodies. ForWestern blot analysis, cells were disrupted with RIPA buffer, boiled ingel loading buffer and separated on a 10% SDS-PAGE gel. Afterelectrophoresis, the proteins were transferred to a PVDF membrane(Millipore). The filter was probed with various antibodies andvisualized with appropriate HRP-conjugated secondary antibodies (SantaCruz Biotechnology) and ECL reagents (Perkin Elmer Life Sciences).

Antibody against ERK1/2 (K-23) was purchased from Santa CruzBiotechnology. Antibodies used to analyze activation of the MAP kinasepathway included the phosphorylated forms of Mek1 and ERK1/2, and werepurchased from Cell Signaling Technology. Rat anti-ER-α antibody (11222)was purchased from Research Diagnostic Inc. Antibodies of COPB (Y-20),mSin3A (AK-11), and 5′ nucleotidase (H-300) were purchased from SantaCruz biotechnology Inc. D4-GDI (clone 97A1015) was obtained from UpstateBiotechnology.

The polyclonal anti-ER-α36 antibody was raised in rabbit against thesynthesized peptide antigen according to the last 15 amino acids at theC-terminal region of ER-α36 that are unique to ER-α36 (Alpha DiagnosticInc.). An affinity column of synthesized peptide used to raise theantibody was used to purify the antibody. The specificity of theantibody has also been tested in ER-α36 expression vector transfectedHEK293 cells that do not express endogenous ER-α36. Immunofluorescenceassay showed that immunoreactive signals of the anti-ER-α36 antibodywere detected only in transient transfectants with ER-α36-expressingvectors but not in transfectants expressing a mutant ER-α36 lacking theC-terminus, suggesting that the ER-α36 antibody is highly specific.

DNA transfection and luciferase assay. For transient transfectionassays, HEK293 cells subseeded in 6 well dishes were grown to 60-70%confluence in phenol-red free medium plus 2.5% steroid-free fetal calfserum. Cells were washed and transiently transfected with total 5 μg ofplasmids (2 μg of the reporter plasmid 2×ERE-tk-Luc together with 1.5 μgof the expression vector pSG5, 1.5 μg of pSG hERα66 or 1.5 μg of pSGhERβ alone or with 1.5 μg of ER-α36 expression vector) with FuGene6reagent (Roche Molecular Biochemicals). A reporter plasmid containingtwo EREs (sequence from −331 to −289 of the chicken Vitellogenin A2gene) placed upstream of the thymidine kinase promoter (2×ERE-tk-Luc,obtained from Dr. Katarine Pettersson, Karolinska Institute, Sweden) wasused. The expression vectors containing ER-α66 and β were also obtainedfrom Dr. Katarine Pettersson. The expression vector of ER-α46 wasobtained from Dr. Zafar Nawaz (Creighton University Medical Center,Omaha, Nebr.). Cells were treated with or without E2 (10 nM) for 12hours before being assayed for luciferase activity. Luciferase assayswere performed using the Luciferase Assay kit from Promega. Valuescorrespond to the average±standard deviation of more than three separatetransfection experiments.

RNA extraction and Northern blot analysis. Total cellular RNA wasisolated using Trizol (Invitrogen), according to the manufacturer'sinstruction. Ten μg of total RNA was separated by electrophoresis on a1.2% formamide/formaldehyde gel and blotted onto a nylon membrane(Hybond-N, Amersham Pharmacia Biotech). The blots were prehybridized for1 hour and hybridized for 2 hours in Quick-Hybridization solution(Amersham Pharmacia Biotech) at 65° C. The probes included a 410 bp cDNAfragment from the 3′ untranslated region of ER-α36 that is unique toER-α36, and a β-actin DNA probe from BD Clonetech. The DNA probes werelabeled with ³²P dCTP and a Rediprime II DNA labeling kit (AmershamBiotech). Blots were autoradiographed using intensifying screens at −70°C. overnight. The same membranes were stripped and reprobed with alabeled β-actin DNA probe to confirm equal loading.

Breast cancer specimens and immunmohistochemistry assay.Paraffin-embedded human breast cancer specimens were obtained from theDepartment of Pathology, the Sir Run Run Shaw Hospital, Hangzhou P. R.China. Immunohistochemical staining was one using UltraSensitive™ S-Pkit (Maixin-Bio, China) according to the manufacturer's instruction,with an ER-α66 specific antibody (LabVision Corporation, USA) and theER-α36 specific antibody as primary antibody, respectively.

Results

To further confirm that ER-α36 is a naturally occurring isoform ofER-α66, Northern blot analysis of total RNA from a normal mammaryepithelial cell line, MCF10A, and ER-positive and -negative (i.e.,ER-α66-positive and − negative) breast cancer cells was performed. A DNAprobe was synthesized using the RT-PCR method with the primer pairsdesigned according to the 3′ untranslated region of the ER-α36 that isunique to ER-α36 (5′-GCAAAGAAGAGAATCCTGAACTTGCATCCT (SEQ ID NO:26) and5′ TTAGTCAGGTATTTAATAACTAGGAATTG (SEQ ID NO:27)). The Northern blotanalysis showed that a single mRNA with estimated size of 5.6 kb wasidentified in ER-positive (i.e., ER-α66-positive) breast cancer cellsMCF7 but not in MCF10A cells (FIG. 13). Surprisingly, ER-α36 was alsoexpressed in MDA-MB-231 cells, a well known ER-negative (i.e.,ER-α66-negative) breast cancer cell line (FIG. 3). This data indicatesthat transcripts with the predicted size of the ER-α36 mRNA areexpressed in breast cancer cells, and even in breast cancer cells thatlack the ER-α66.

ER-α36 inhibits transactivation activities of liganded- andunliganded-ER-α66 and -β. We first tested whether ER-α36 that lacks bothAF-1 and AF-2 domains retains any transcriptional activity. Transienttransfection assays were performed in HEK293 cells using aluciferase-expressing reporter construct that contains two estrogenresponse elements (ERE) placed upstream of the thymidine kinase promoter(2×ERE-tk-Luc). The HEK293 cell line was selected since it was reportedpreviously that the AF-1 and −2 of ER-α66 function equally well inHEK293 cells (Denger et al., Mol. Endocrinol., 15, 2064-2077 (2001)). Asshown in FIG. 14, we found that ER-α36 exhibits no intrinsictranscriptional activity in the presence and absence of E2β, consistentwith the finding that ER-α36 lacks both transcription activationdomains. We then assessed the regulatory function of ER-α36 intranscriptional transactivation activities mediated by the AF-1 and -2domains of the ER-α66. Co-expression of ER-α36 strongly inhibited thetransactivation activity of ER-α66 in the presence and absence of E2β(FIG. 14), suggesting that ER-α36 inhibits the transactivationactivities mediated by the AF-1 and AF-2 domains of ER-α66. Furthermore,ER-α36 also inhibited ligand-dependent and -independent transactivationactivities of ER-β (FIG. 14).

ER-α36 mediates membrane-initiated estrogen signaling pathway. Previousreports have indicated that E2β stimulates a rapid activation of theMAPK/ERK pathway (Razandi et al., Mol. Endocrinol., 13, 307-319 (1999),Watters et al., Endocrinol., 138, 4030-4033 (1997), and Migliaccio etal., EMBO J., 15, 1292-1300 (1996)). To determine whether ER-α36 isinvolved in this signaling pathway, we established stable cells thatexpress exogenous ER-α36 in HEK293 cells that do not express endogenousER-α. Whole ER-α36 transfected HEK293 cells were incubated withfluorescein isothiocyanate (FITC)-conjugated E2β-BSA (E2β-BSA-FITC) thatis membrane impermeable. The cell surface of the ER-α36 transfectedcells was strongly labeled by the E2β-BSA-FTIC whereas the control cellstransfected with empty vector were not labeled by E2β-BSA-FITC. Celllysates were prepared from quiescent cells that were either untreated ortreated with E2β (10 nM) for various lengths of time. ERK activation wasmeasured by immunoblotting using phosphorylation state-dependent and-independent antibodies. A 10-fold increase in the phosphorylation ofERK1/2 that lasted about 45 minutes was observed within 5 minutes in thecells transfected with ER-α36 expression vector but not in the controlcells transfected with empty vector (FIGS. 15 a and 15 b). However,serum (20% for 10 minutes) was able to activate ERK1/2 in these controlcells (FIG. 15 b), indicating that there is no global defect of the MAPKsignaling pathway in these cells. Furthermore, Mek1, the kinase thatphosphorylates and activates ERK1/2, is also activated in response toE2β in ER-α36 transfected cells (FIG. 15 a). To provide further evidencefor activation of the ERK1/2 by a membrane-initiated estrogen signaling,ER-α36 transfected cells were also treated with E2β-BSA, a membraneimpermeable form of E2β. A strong activation of ERK1/2 phosphorylationwas also observed in E2β-BSA treated cells (FIG. 15 a).

ER-α36 mediates activation of the MAPK signaling pathway stimulated bydifferent estrogens and anti-estrogens. We also treated ER-α36transfected cells for 10 minutes with estrone (E1), 17β-estradiol (E2β),17α-estradiol (E2α), estriol (E3), or estetrol (E4), and found that allof these estrogens except E1 activated ERK1/2 phosphorylation at a verysimilar level, suggesting ER-α36 may recognize these estrogens at asimilar level (FIG. 15 c). We then included the anti-estrogens includingTamoxifen, 40H-Tamoxifen, ICI-182, 780 in the experiment to test whetherER-α36 mediated estrogen signaling is sensitive to anti-estrogens.Tamoxifen, 4OH-Tamoxifen and the pure anti-estrogen ICI-182, 780 did notblock ERK1/2 activation mediated by ER-α36. On the contrary, the effectsare even stronger compared to the effects mediated by E2β alone (FIG. 15c). When ER-α36 transfected cells were treated with 1 μM Tamoxifenalone, a concentration that can blunt both ER-α66 and 13, a strong andpersistent ERK1/2 activation that lasted longer than eight hours wasobserved (FIG. 5 d). Tamoxifen at the same concentration, however, hadno effect in control 293 cells transfected with empty expression vector.

ER-α36 mediates E2-stimulated cell proliferation. To further determinewhether the estrogen activated MAPK pathway mediated by ER-α36 can leadto the transcriptional signaling in cell nucleus, we examined theability of membrane-initiated estrogen signaling to activate thetranscription factor Elk, a downstream effector of the MAPK/ERKsignaling pathway. We transiently transfected ER-α36 expressing 293cells with the ERK-responsive GAL-Elk chimeric transcription factor,consisting of the DNA-binding domain of yeast transcription factor GAL4fused to the ERK-responsive trans-activation domain of human Elk1, andmeasured its activity in vivo on the expression of a GAL-Elk bindingreporter gene in the presence of E2β. The reporter gene was 5×Gal4-LUC,a luciferase reporter plasmid containing five Gal4 DNA binding sites. Abacteria β-galactosidase expression vector was used to controltransfection efficiency. After transfection, the cell culture wasmaintained in estrogen free medium for 36 hours before E2β (10 nM) wasadded for 12 hours. Luciferase activities with standard deviation arerepresentative of more than three experiments performed in duplicates.Estrogen treatment of ER-α36 transfected cells induced about two-foldincrease of Elk/Gal4 fusion protein-mediated trans-activation of thereporter whereas E2P had no effect on the transcription activity of theElk/Gal4 fusion protein in the control cells transfected with emptyvector (FIG. 16 a).

We next addressed whether the ER-α36 can mediate estrogen-stimulatedcell proliferation. Proliferation of the ER-α36 transfected cells andcontrol cells in the presence and absence of E2β was evaluated by theMTT assay. Proliferation of ER-α36 transfected cells was stimulated byE2β treatment while E2β had no effect on the growth of the control cellstransfected with empty expression vector (FIG. 16 b). The inclusion ofthe anti-estrogens including Tamoxifen and 4OH-Tamoxifen did not blockE2β-stimulated cell growth (FIG. 16 b). Tamoxifen or 4OH-Tamoxifen alonestrongly stimulated growth of the ER-α36 transfected cells. However, thespecific inhibitor of MAPK pathway, UO126, strongly inhibitedE2β-stimulated cell growth. These data suggest that ER-α36-mediatedmembrane estrogen signaling stimulates cell growth through activation ofthe MAPK/ERK signaling pathway. The data also suggest anti-estrogensalso stimulate cell growth through ER-α36.

ER-α36 is predominantly a membrane-based estrogen receptor. To furthercharacterize ER-α36, we have successfully developed a polyclonalanti-ER-α36 antibody raised against the 15 amino acids at the C-terminalregion of ER-α36 that are unique to ER-α36. An affinity column ofsynthesized peptide used to raise the antibody was used to purify theantibody. Western blot analysis of proteins prepared from normal mammaryepithelial cells and established breast cancer cell lines using thisantibody demonstrated a single protein band with 37-kDa molecular weightin some breast cancer cells but not in normal mammary epithelial cells(FIG. 17 a). ER-α36 is expressed in MDA-MB-231, MDA-MB436 and HB3396cells, three well-known ER-α66 negative breast cancer cell lines, andalso is expressed in ER-α66 positive breast cancer cells MCF7 but not inT47D (FIG. 17 a), consistent with our Northern blot data that ER-α36 isexpressed in ER-α66 negative breast cancer cells. To evaluate furtherthe possibility that ER-α36 is expressed in ER-α66 negative breastcancer cells, indirect immunofluorescent assay and confocal microscopyin permeabilized MDA-MB-231 cells using the anti-ER-α36 specificantibody showed that ER-α36 is expressed on the plasma membrane,cytoplasm and nucleus of the ER-α66 negative breast cancer cells,MDA-MB-231.

To further assess the ER-α36 compartmentalization in cells, thesubcellular fractionation assay was performed to isolate nuclei, plasmamembranes, and cytosol from ER-α36 transfected HEK293 cells. ER-α36 wasidentified by immunodetection from the different fractions. A highpercentage of ER-α36 (˜50%) was localized on the plasma membrane and alow percentage of it in cytosol (˜40%) and nucleus (˜10%). To excludecross-contamination of different fractions, the fraction purity wasexamined by Western blot analysis with different marker proteinsincluding mSin3A (nucleus), GDP dissociation inhibitor (cytosol), 5′nucleotidase (plasma membrane), and β-COP (Golgi). These resultsconfirmed that there was no contamination among different fractions.This experiment established that ER-α36 is predominantly amembrane-based estrogen receptor (FIG. 17 b).

ER-α36 is expressed in ER-α66 negative breast cancer specimens. Tofurther determine the relevance of ER-α36 to human breast cancer, weexamined the ER-α36 expression patterns in human breast cancer specimenswith an immunohistochemistry assay using the specific anti-ER-α36antibody. In situ analysis of human breast tissue showed theup-regulation of ER-α36 in breast cancer. Cells that were positive forER protein stained brown, and nuclei were stained blue with hematoxylin.Among 35 cases of breast cancer specimens examined, 21 of them (60%)stained positive for ER-α36, and 21 of them (60%) positive for ER-04.66(Table 2). Consistent with our Northern and Western blot analyses, 11out of 14 (78%) breast cancer specimens that stained negative for ER-α66were stained positive for ER-α36, indicating most ER-negative (i.e.,ER-α66-negative) breast cancers still express ER-α36. In situ stainingof ER-α36 in a normal section of tissue found in a human ductalcarcinoma showed ER-α36 expression only in lumenal epithelial cells andmainly localized in cytoplasm and plasma membrane. Tumor sections werefrom human infiltrating ductal carcinoma and from invasive ductalcarcinoma. All 21 cases ER-α36 positive specimens exhibited ER-α36immunostaining patterns predominantly outside of the cell nucleus,contrary to the mainly nuclear staining of ER-α66. Like ER-α66, someluminal epithelial cells in neighboring normal tissue were also stainedpositive for ER-α36. These results demonstrate that like ER-α66, ER-α36is expressed in two-thirds of the human breast cancer examined, andsuggest that ER-α36 may be involved in development of ER-α66-negativebreast cancer.

TABLE 2 ER-α36 and 66 expression: Survey of immunostaining in humanbreast cancer. Case# ER-α66 ER-α36 Case# ER-α66 ER-α36 04-06108D +++ +02-09537B +++ − 04-06278G +++ + 03-22792D ++ − 03-19482D +++ ++04-10881D + − 03-13610E +++ + 03-10071F − ++ 01-09182B ++ + 03-22971M− + 02-17950B ++ + 01-08119D − +/− 02-07748H ++ + 02-02018D − +01-13537G ++ + 02-04567E − + 00-0319D ++ +/− 04-07055E − + 03-04069G ++/− 03-05946C − + 00-02787D ++ − 03-22586I − + 02-18513F ++ − 01-02877A− + 04-08474J ++ − 01-17570C − +/− 02-01265M ++ − 00-08489G − +/−03-07870E + − 00-02202F − − 02-04206F ++ − 98-03898D − − 02-12985F +++ −03-04898B − − 03-08862G ++ −

In this study, a novel variant of ER-α, ER-α36, has been identified,cloned and characterized. This ER-α isoform is the product of atranscript initiated from a previous unidentified promoter in the firstintron of ER-α66 gene. The putative promoter region of the ER-α36contains a TATA binding protein (TBP) recognition sequence upstream ofthe ER-α36 cDNA start site, and several Sp1, NF-kB and Ap1 binding sites(FIG. 12). We have cloned the 5′ flanking region of ER-α36 and confirmedthat it possess strong promoter activity. Furthermore, a perfect halfERE site was identified at the 5′ flanking region of ER-α36, suggestingthat ER-α36 is subjected to ER-mediated transcriptional regulation.

ER-α36 protein is identical to the ER-α66 protein encoded by exons 2-6of the ER-α66 gene. This isoform is devoid of the domains previouslyidentified to have transactivation activities, AF-1 and -2. Indeed,analysis of ER-α36 transactivation activity demonstrated that ER-α36lacks intrinsic transcriptional activity. However, ER-α36 efficientlysuppresses the transactivation activities mediated by the AF-1 and -2domains of liganded- and unliganded-ER-α66 and -β, indicating thatER-α36 is a potent inhibitor of the genomic estrogen signaling. Thisfinding parallels the previous report that ER-α46 that lacks the AF-1domain functions as a powerful competitor to suppress the AF-1 activityof the ER-α66.

The presence of a plasma membrane-based ER that triggers rapid estrogensignaling was controversial for a long time, since the molecularidentity of this receptor has not been established. Previously, Razandiusing transfection assay reported that both ER-α66 and -β can initiatemembrane estrogen signaling, although only a very small percentage ofthem was expressed on the cell surface (Razandi et al., Mol.Endocrinol., 13, 307-319 (1999)), suggesting that these ERs may beinvolved in membrane-initiated estrogen signaling in addition to theirtraditional roles in genomic estrogen signaling. Recently, the 46-kDaisoform of ER-α was localized on cell surface and found to mediateestrogen-stimulated eNOS phosphorylation (Li et al., Pro. Natl. Acad.Sci. USA, 100, 4807-4812 (2003)). Here, we demonstrated that anotherER-α variant, ER-α36, is predominantly localized on the plasma membraneand mediates activation of the MAPK/ERK pathway induced bymembrane-initiated estrogen signaling. Moreover, since ER-α36 totallylacks intrinsic transactivation activity and only functions as aregulator of genomic estrogen signaling, ER-α36 may act primarily as amembrane-based estrogen receptor to mediate membrane-initiated estrogensignaling. Previously, it has been reported that some E2β-mediated rapidactions occur in neurons of ER-α gene knockout (aERKO) mice and theseactions are not blocked by ICI 182,780 (Gu et al., Endocrinology, 140,660-666 (1999)), suggesting that the existence of more than onemembrane-initiated estrogen signaling pathway. We demonstrated here thatanti-estrogens did not block ER-α36-mediated MAPK/ERK activation,suggesting that ER-α36 is involved in the anti-estrogen insensitivesignaling pathway previously described. As αERKO mice were created by aninsertional disruption of the first coding exon of the mouse ER-α gene(the exon that is skipped in the generation of transcripts of ER-α36),it is likely that the production of the mouse counterpart of ER-α36remains normal in these knockout mice. Thus, ER-α36 may contribute tothe remaining estrogen effects observed in these mice. Recently,Toran-Allerand et al. (J. Neuroscience 22, 8391-8401 (2002)) reportedthe existence of a novel plasma membrane-associated estrogen receptor(ER-X) with an estimated molecular weight of 63-65 kDa. ER-X shows somesimilarities with ER-α36, such as reacting with antibodies to theligand-binding domain of ER-α66 and responding equally well to E2α andβ. However, the molecular identity of these two receptors awaits for thecloning and sequencing of ER-X.

We have also shown that ER-α36 promotes membrane-initiated activation ofthe MAPK/ERK pathway that leads to estrogen-stimulated cellproliferation. Thus, ER-α36, that is devoid of intrinsic transcriptionactivity, is sufficient to promote estrogen-stimulated cell growth,offering support to the previous report that a transcriptionallyinactive mutant of ER-α66 induces DNA synthesis. These data togethersuggest that transcriptional activities of ER may not be required topromote estrogen-stimulated cell growth. Surprisingly, we also observedthat anti-estrogens such as Tamoxifen strongly activated the MAPK/ERKsignaling and stimulated cell growth during the experiment period (48hours). This finding is in good agreement with the idea that Tamoxifenfunctions as both agonist and antagonist of estrogen signaling, andsuggests that ER-α36 may be also involved in membrane-initiatedanti-estrogen signaling.

It is well known that breast cancer cells with an ER-α66 positivephenotype (ER-positive breast cancer) are more differentiated and havelower metastatic potential than ER-α-negative tumors (McGuire, W. L.Prognostic factors in primary breast cancer. Cancer Surv. 5, 527-536(1986)). It is interesting that ER-α36 is expressed not only in thesubset of ER-α66-positive breast cancers but also in most ER-α66negative breast cancers examined. Corroborating these results, it hasbeen reported that estrogen signaling induced a rapid activation of thePI3K/Akt pathway in MDA-MB-231 cells that could not be blocked byestrogen antagonists (Tsai et al., Cancer Res. 61, 8390-8392 (2001)),which was explained as estrogen signaling through an ER-independentpathway. High concentration of Tamoxifen also has been shown to induceapoptosis in MDA-MB-231 cells (Mandleker et al., Apoptosis 6, 469-477(2001)). These data strongly suggest that ER-α66-negative breast cancermay still retain estrogen or anti-estrogen effects mediated bymembrane-initiated signaling.

ER-α36 also possess a unique ligand-binding domain by replacing the last5 helixes (helix 8-12) of the 12 helixes in the ER-α66 with a unique 27amino acid domain, which may change the ligand-binding specificity andaffinity of ER-α36. Indeed, we found that ER-α36 elicitedmembrane-initiated signaling equally well in response to E2α and β, E3and E4. Thus, ER-α36 appears to possess a much broader ligand-bindingspectrum than ER-α66, which makes ER-α36 potentially a more potentmediator of mitogenic signaling. Further analysis of the ligand-bindingspecificity and affinity of ER-α36 will help design anti-estrogensspecific for ER-α36 that can be used to treat ER-α negative (i.e.,ER-α66-negative) breast cancer.

Example 4 E2β Promotes Growth of ER-α66 Negative MDA-MB-231 Cells inSoft Agar

To determine anchorage-independent growth in soft-agar in the presenceand absence of E2β and tamoxifen together or separately, five hundredMDA-MB-231 cells were suspended in 3 ml of 3.5% (wt/vol) agar containingphenol red-free DMEM/F12 medium plus 10% E2-free fetal calf serum. Thecells were then overlaid onto a 0.7% (wt/vol) agar containing phenol-redfree DMEM/F12 medium plus 10% E2-free fetal calf serum in five replica60 mm dishes. Cells on soft agar were covered with medium plus 10% E2free fetal calf serum with or without 1 nM E2β, or combined with 1 nMtamoxifen. After three weeks, colonies were scored using an invertedmicroscope. As seen in FIG. 18, we found that E2 treatment stronglypromoted anchorage-independent growth of ER-α66 negative MDA-MB-231cells in soft agar, whereas anti-estrogen Tamoxifen inhibits the effectof E2β, indicating that ER-α66 negative MDA-MB-231 cells retainresponsiveness to estrogen signaling presumably through Er-α36.

Example 5 E2β Induces Membrane-Initiated Estrogen Signaling in ER-α66Negative MDA-MB-231 Cells

To determine whether E2β induces membrane initiated estrogen signalingin ER-α66 negative MDA-MB-231, serum starved MDA-MB-231 cells weretreated with 1 nM E2β for different time periods. For Western blotanalysis, cells were disrupted with RIPA buffer, boiled in gel loadingbuffer and separated on a 10% SDS-PAGE gel. After electrophoresis, theproteins were transferred to a PVDF membrane (Millipore). The filter wasprobed with antibody against ERK1/2 (K-23) (Santa Cruz Biotechnology),or antibody used against the

1. A cell comprising an exogenous coding region, wherein the codingregion encodes a first polypeptide comprising SEQ ID NO:20 or a secondpolypeptide comprising an amino acid sequence having at least 90%identity to SEQ ID NO:20, wherein the second polypeptide has ER-α36activity.
 2. The cell of claim 1 wherein the coding region is operablylinked to a constitutive promoter.
 3. The cell of claim 1 wherein thecell is a eukaryotic cell.
 4. The cell of claim 1 wherein the cell is aprokaryotic cell.
 5. A cell expressing an exogenous polypeptide, whereinthe polypeptide comprises SEQ ID NO:20, or comprises an amino acidsequence having at least 90% identity to SEQ ID NO:20 and has ER-α36activity.
 6. The cell of claim 5 wherein the coding region is operablylinked to a constitutive promoter.
 7. The cell of claim 5 wherein thecell is a eukaryotic cell.
 8. The cell of claim 5 wherein the cell is aprokaryotic cell.